P2X3 receptor antagonists for treatment of pain

ABSTRACT

The subject invention relates to novel P2X 3  receptor antagonists that play a critical role in treating disease states associated with pain, in particular peripheral pain, inflammatory pain, or tissue injury pain that can be treated using a P2X 3  receptor subunit modulator.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/US2010/027303 filed on Mar. 15, 2010, which claims the benefit under35 U.S.C. 119(e) of U.S. Provisional Application No. 61/210,778, filedMar. 23, 2009.

FIELD OF THE INVENTION

The invention relates generally to compounds which act as modulators,e.g., antagonists of the P2X₃ receptor, compositions and therapeuticuses thereof.

BACKGROUND OF THE INVENTION

Purines, acting via an extracellular purinoreceptor, have beenimplicated as having a variety of physiological and pathological roles.(See, Burnstock (1993) Drug Dev. Res. 28:195-206.) Purinoreceptors (P2)have been generally categorized as either metabotropic nucleotidereceptors or ionotropic receptors for extracellular nucleotides.Metabotropic nucleotide receptors (usually designated P2Y or P2Y_((n)),where “n” is a subscript integer indicating subtype) are believed todiffer from ionotropic receptors (usually designated P2X or P2X_((n)) inthat they are based on a different fundamental means of transmembranesignal transduction: P2Y receptors operate through a G protein-coupledsystem, while P2X receptors are ligand-gated ion channels.

At least seven P2X receptors, and the cDNA sequences encoding them, havebeen identified to date. P2X₁ cDNA was cloned from the smooth muscle ofthe rat vas deferens (Valera et al. (1994) Nature 371:516-519) and P2X₂cDNA was cloned from PC12 cells (Brake et al. (1994) Nature371:519-523). Five other P2X receptors have been found in cDNA librariesby virtue of their sequence similarity to P2X₁ and P2X₂—P2X₃: Lewis etal. (1995) Nature 377:432-435, Chen et al. (1995) Nature 377:428-431;P2X₁: Buell et al. (1996) EMBO J. 15:55-62, Seguela et al. (1996) J.Neurosci. 16:448-455, Bo et al. (1995) FEBS Lett. 375:129-133, Soto etal. (1996) Proc. Natl. Acad. Sci. USA 93:3684-3688, Wang et al. (1996)Biochem. Biophys. Res. Commun. 220:196-202; P2X₅: Cello et al. (1996) J.Neurosci. 16:2495-2507, Garcia-Guzman et al. (1996) FEBS Lett.388:123-127; P2X₆: Cello et al. (1996), supra, Soto et al. (1996)Biochem. Biophys. Res. Commun. 223:456-460; P2X₂: Surprenant et al.(1996) Science 272:735-738). For a comparison of the amino acidsequences of rat P2X receptor see Buell et al. (1996) Eur. J. Neurosci.8:2221-2228.

Purinergic receptors, in particular, P2X receptors, are known tofunction as homomultimeric cation-permeable ion channels and, in somecases, as heteromeric channels consisting of two different P2X receptorsubtypes (Lewis et al., Nature 377:432-435 (1995); Le et al., J.Neurosci. 18:7152-7159 (1998); Torres et al., Mol. Pharmacol. 54:989-993(1998)). The P2X₂ and P2X₃ subunits form functional channels whenexpressed alone, and can also form a functional heteromultimeric channelthat has properties similar to currents seen in native sensory channelswhen co-expressed. At least one pair of P2X receptor subtypes, P2X₂ andP2X₃, functions as a heteromeric channel in rat nodose ganglion neuronswhere it exhibits distinct pharmacological and electrophysiologicalproperties (Lewis et al., supra (1995)).

Native P2X receptors are known to form rapidly activated, nonselectivecationic channels upon activation by ATP. The channels formed by P2Xreceptors generally have high Ca²⁺ permeability (P_((Ca)))/P_((Na))).With respect to individual receptors, the P2X₃ purinergic receptor is aligand-gated cation channel that is selectively permeable to smallcations. Known ligands for P2X receptors include natural nucleotides,for example, ATP, UTP, UDP, or synthetic nucleotides, for example2-methylthioATP. ATP, in addition to its function as an intracellularenergy donor, is now recognized as an important neurotransmitter orcotransmitter, in both the central and peripheral nervous system(Ralevic, V., et al., Pharmacol. Rev., 50:413-492 (1998)). It isreleased from a variety of cell types, including nerve fibers, uponstimulation and produces diverse effects on many tissues by activationof specific membrane receptors including purinoreceptors (P2 receptor)(See Burnstock, G., Pharmacol. Rev., 24:509-581 (1972); Burnstock, G.,Cell Membrane Receptor for Drugs and Hormones: A MultidisciplinaryApproach, edited by R. W. Straub and L. Bolid. New York: Raven, 1978, p.107-118). With respect to the P2X purinergic receptor, data suggest thatATP is capable of activating P2X₃ homomeric receptors and P2X₂/P2X₃heteromeric receptors where it functions as an excitatoryneurotransmitter in the spinal cord dorsal horn and in primary afferentsfrom sensory ganglia. In vitro, co-expression of P2X₂ and P2X₃ receptorsubunits is necessary to produce ATP-gated currents with the propertiesseen in some sensory neurons. See, Lewis, et al. (1995) Nature377:432-435.

ATP, and to a lesser extent, adenosine, can stimulate sensory nerveendings resulting in intense pain and a pronounced increase in sensorynerve discharge. According to available data, ATP released from damagedcells can evoke pain by activating P2X₃ homomeric receptors, orP2X₂/P2X₃ heteromeric receptors expressed on nociceptive nerve endingsof sensory nerves. This is consistent with reports of the induction ofpain by intradermally applied ATP in the human blister-base model; theidentification of P2X₃ containing receptor on nociceptive neurons in thetooth pulp; and with reports that P2X antagonists are analgesic inanimal models. To date, research data suggests that the mechanismwhereby ATP-induced activation of the P2X purinergic receptors on dorsalroot ganglion nerve terminals in the spinal cord and on neurons in thebrain results in pain sensation is by the stimulation of the release ofglutamate, a key neurotransmitter involved in nociceptive signaling.

It has also been recently demonstrated that P2X₃ receptor genedisruption results in a diminished sensitivity to noxious chemicalstimuli and reduced pain. The nociceptive effects of exogenouslyadministered ATP and P2X containing receptor agonists have also beendemonstrated in laboratory animals. See Bland-Ward et al., Dr. J.Pharmacol. 122:366-371 (1997); Hamilton et al., Br. J. Phamacol.126:326-332 (1999). The peripheral nociceptive actions of P2X activationand stimulation of spinal P2X containing receptor also contribute tonociception as indicated by the ability of intrathecally (i.t.)administered P2 receptor agonists to increase sensitivity to acute andpersistent noxious stimuli in rodents. See Driessen et al., Brain Res.666:182-188 (1994); Tsuda et al., Br. J. Pharmacol, 127:449-4S6 (1999);Tsuda et al., Br. J. Pharmacol. 128:1497-1504 (1999). A selective P2receptor-mediated increase in ectopic neuronal excitability that islocalized to damaged sensory afferents has also been recently reportedin rats following chronic constriction nerve injury. See Chen et al.,NeuroReport 10:2779-2782 (1999). This role in pain transmission isconsistent with the observation that the rat P2X₃ receptor expression isfound primarily in a subset of neurons of the sensory ganglia, which areinvolved in pain transmission. See Chen et al., Nature 377:428-430(1995); Vulchanova et al., Neuropharmacol. 36:1229-1242 (1997). See alsoUS20080004442, US200700409609, WO2007041087, WO2006119504, WO200112627,WO2007001973, U.S. Ser. No. 61/132,178, U.S. Ser. No. 61/001,376, U.S.Ser. No. 61/197,869 and WO2007010553.

Taken together, the functional and immunohistochemical localization ofP2X₃ containing receptors (P2X₃ and/or P2X₂₁₃) on sensory nervesindicates that these P2X receptors may have a primary role in mediatingthe nociceptive effects of ATP. Thus, compounds which block or inhibitactivation of P2X₃ receptors serve to block the pain stimulus. More,receptor antagonists to compounds which normally activate the P2X₃receptor and/or P2X₂/P2X₃ heteromeric channels, such as ATP, couldsuccessfully block the transmission of pain. Indeed, modulators of P2Xreceptors, e.g., P2X₃ receptor may find use as analgesics.

Additionally, compounds that block or inhibit activation of P2X₃receptors also serve to treat genitourinary, gastrointestinal andrespiratory diseases, conditions and disorders or receptor antagoniststo compounds which normally activate the P2X₃ receptor and/or P2X₂/P2X₃heteromeric channels, such as ATP are useful for treatment ofgenitourinary, gastrointestinal and respiratory diseases, conditions anddisorders.

Burnstock (1999) J. Anatomy 194:335-342; and Ferguson et al. (1997) J.Physiol. 505:503-511 disclose that P2X receptor subunits have been foundon afferents in rodent and human bladder urothelium. There data suggeststhat ATP may be released from epithelial/endothelial cells of theurinary bladder or other hollow organs as a result of distention. ATPreleased in this manner may serve a role in conveying information tosensory neurons located in subepithelial components, e.g., suburotheliallamina propria (Namasibayam, et al. (1999) BJU Intl. 84:854-860), P2Xreceptors have been studied in a number of neurons including sensory,sympathetic, parasympathetic, mesenteric, and central neurons (Zhong, etal. (1998) Br. J. Pharmacol. 125:771-781). These studies indicate thatpurinergic receptors play a role in affterent neurotransmission from thebladder, and that modulators of P2X receptors are potentially useful inthe treatment of bladder disorders such as urinary incontinence andother genitourinary diseases or conditions.

P2X₃ receptors have been shown to be expressed in human colon, and areexpressed at higher levels in inflamed colon, than in normal colon (Y.Yiangou et al, Neurokastroenterol Mot (2001) 13:365-69). P2X₃ receptorshave also been implicated in detection of distension or intraluminalpressure in the intestine and initiation of reflex contractions (X. Bianet al. J. Physiol (2003) 551.1:309-22), and have linked this to coilitis(G. Wynn et al., Am J. Physiol Gastrointest Liver Physiol (2004)287:G647-57). P2X₃ receptors also have been shown to be expressed inpulmonary neuroepithelial bodies (NEBs), implicating the receptor inpain transmission in the lung (Inge Brouns et al., Am J. Respir Cell MolBiol (2000) 23:52061). Additionally, P2X₂ and P2X₃ receptors have beenimplicated in pO₂ detection in pulmonary NEBs (W. Rong et al., J.Neurosci (2003) 23(36):11315-21).

However, the utility of available purinergic ligands to evaluate therole of individual P2 receptor subtypes in mammalian physiology has beencomplicated by the susceptibility of P2 receptor agonists to undergoenzymatic degradation. As well, the study of the role of an individualP2X receptor is hampered by the lack of receptor subtype-specificagonists and antagonists.

Consequently, the state of the art begs an inquiry into methods and/orcompounds which will provide the ability to regulate or control the P2Xreceptors, for example, P2X₃, because control of such receptors willprovide the ability to minimize pain in patients in need of suchtreatment. In addition, for both research and therapeutic purposes thereis a need in the art for specific agonists and antagonists for each P2Xreceptor subtype and, in particular, agents that will be effective invivo, as well as for methods for identifying purinoreceptor-specificagonist and antagonist compounds.

The present invention aims to overcome some of the aforementioneddrawbacks by providing novel P2X₃ receptor antagonists that play acritical role in treating disease states associated with pain, inparticular peripheral pain, inflammatory pain, or tissue injury painthat can be treated using a P2X₃ receptor subunit modulator.

SUMMARY OF THE INVENTION

The present invention relates to a novel P2X₃ type receptor antagonistsof structural formula I:

or pharmaceutically acceptable salts and individual enantiomers anddiastereomers thereof wherein: A represents thienopyrazolyl, indazolyl,indolyl, or regioisomers thereof;B represents, a bond, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₃₋₁₀ cycloalkyl, C₅₋₁₀heterocyclyl, or C₆₋₁₀ aryl;R¹ represents H, C₁₋₆ alkyl, halogen, (CH₂)_(n)CF₃, C₃₋₁₀ cycloalkyl,C(R²)₂OH, —O—, CN, (CH₂)_(n)OR², NHC(O)R², (CH₂)_(n)C₅₋₁₀ heterocyclyl,(CH₂)_(n)C₆₋₁₀ aryl, or C₁₋₆ alkoxy; said alkyl, cycloalkyl,heterocyclyl and aryl optionally substituted with 1 to 3 groups of C₁₋₆alkyl, halogen, hydroxyl, (CH₂)_(n)CF₃, or CN;R² represents H, C₁₋₆ alkyl, CF₃, OH;R³ represents CR²R⁴R⁵, (CHR²)_(n)C₃₋₁₀ cycloalkyl, (CHR²)_(n)C₆₋₁₀ aryl,(CHR²)_(n)C₅₋₁₀ heterocycle, said cycloalkyl, aryl and heterocyclyloptionally substituted with 1 to 3 groups of R^(a);or R² and R³ can be combined with the nitrogen to which they areattached to form a C₅₋₁₀ heterocyclyl optionally substituted with 1 to 3groups of R^(a);R⁴ and R⁵ independently represent H, (CH₂)_(n)OR², CHF₂, (CH₂)_(n)C₅₋₁₀heterocyclyl, (CH₂)_(n)C₆₋₁₀ aryl, C₃₋₁₀ cycloalkyl, C₁₋₆ alkoxy, C₂₋₆alkenyl, CF₃, CF₂, C(O)₁₋₂R², or C₁₋₆ alkyl; said alkyl, cycloalkyl,heterocyclyl and aryl optionally substituted with 1 to 3 groups ofR^(a);R⁶ represents hydrogen, OR², (CH₂)_(n)CF₃, halogen, C(R²)₂OR², C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ cycloalkyl, (CH₂)_(n)C₆₋₁₀aryl, (CH₂)_(n)C₅₋₁₀ heterocyclyl, said alkyl, cycloalkyl, aryl andheterocyclyl optionally substituted with 1 to 3 groups of R^(a);R^(a) represents C₁₋₆ alkyl, halogen, hydroxyl, OR² (CH₂)_(n)CF₃, —O—,C₃₋₆ cycloalkyl, NR²C(O)R², C(O)N(R²)₂, C(R²)₂OR², C(O)R²,O(CH₂)_(n)C(O)N(R²)₂, O(CH₂)_(n)C(O)OR², C(O)C₅₋₁₀ heterocyclyl, NO₂,CN, N(R²)₂, C(O)OR², SO₂R², OR², (CH₂)_(n)C₅₋₁₀ heterocyclyl, or(CH₂)_(n)C₆₋₁₀ aryl, said alkyl, heterocyclyl and aryl optionallysubstituted with 1 to 3 groups of C₁₋₆ alkyl, halogen, hydroxyl,(CH₂)_(n)CF₃, or CN; andn represents 0 to 4.

This invention also relates to compositions and methods for using thecompounds disclosed herein. These and other embodiments of the presentinvention will readily occur to those of ordinary skill in the art inview of the disclosure herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel P2X₃ type receptor antagonistsof structural formula I that are useful in treating pain and diseasesassociated with pain.

An embodiment of the present invention is realized when B is a C₆₋₁₀ aryselected from the group consisting of phenyl, or napthyl, preferablyphenyl, said phenyl or napthyl optionally substituted with 1 to 3 groupsof R^(a).

An embodiment of the present invention is realized when B is a C₅₋₁₀heterocyclyl selected from the group consisting of pryidyl, morpholinyl,pyrazinyl, piperonyl, pyrazolyl, thiophenyl, pyrimidinyl, indolyl, or(uranyl, preferably pyridyl, or thiophenyl, all of which optionallysubstituted with 1 to 3 groups of R^(a). A subembodiment of thisinvention is realized when B is pyridyl optionally substituted with 1 to3 groups of R^(a) and all other variables are as previously described.

Another embodiment of the present invention is realized when B is phenyloptionally substituted with 1 to 3 groups of R^(a) and all othervariables are as previously described.

Another embodiment of the present invention is realized when B is C₁₋₆alkyl optionally substituted with 1 to 3 groups of R^(a) and all othervariables are as previously described.

Another embodiment of this present invention is realized when B is abond and all other variables are as originally described.

Yet another embodiment of the present invention is realized when B isC₃₋₁₀ cycloalkyl optionally substituted with 1 to 3 groups of R^(a) andall other variables are as originally described.

Another embodiment of the present invention is realized when A isthienopyrazolyl and all other variables are as previously described. Asubembodiment of this invention is realized when —C(O)NR²R³ is attachedto a carbon atom on A. Another subembodiment of this invention isrealized when B is attached to a carbon atom on the pyrazolyl portion ofthe thienopyrazolyl ring structure.

Another embodiment of the present invention is realized when A isindazolyl and all other variables are as previously described. Asubembodiment of this invention is realized when —C(O)NR²R³ is attachedto a carbon atom on A. Another subembodiment of this invention isrealized when B is attached to a carbon atom on the pyrazolyl containingportion of the indazolyl ring structure.

Another embodiment of the present invention is realized when A isindolyl and all other variables are as previously described. Asubembodiment of this invention is realized when —C(O)NR²R³ is attachedto a carbon atom on A. Another subembodiment of this invention isrealized when B is attached to a carbon atom on the benzyl portion ofthe indolyl ring structure. Still another sub-embodiment of thisinvention is realized when B is attached to a carbon atom on the pyrroleportion of the indolyl ring structure.

Still another embodiment of the invention is realized when R⁶ representshydrogen, C₁₋₆ alkyl, (CH₂)_(n)C₆₋₁₀ aryl, (CH₂)_(n) C₅₋₁₀ heterocyclyl,said alkyl, heterocyclyl and aryl optionally substituted with 1 to 3groups of R^(a), and all other variables are as previously described. Asub-embodiment of this invention is realized when R⁶ is optionallysubstituted C₁₋₆ alkyl, or (CH₂)_(n)phenyl. Another sub-embodiment ofthis invention is realized wherein when R⁶ is a heterocyclyl it isselected from the group consisting of (CHR^(y))_(n)pyridyl,(CHR^(y))_(n)thiophenyl, (CHR^(y))_(n)pyrimidinyl, (CHR^(y))_(n)furanyl,(CHR^(y))_(n)thiadiazolyl, (CHR^(y))_(n)thiazolyl,(CHR^(y))_(n)pyrazolyl, (CHR^(y))_(n)oxadiazolyl,(CHR^(y))_(n)thiazolyl, wherein R^(y) represents H, C₁₋₆ alkyl, CF₃, OH,said alkyl optionally substituted with 1 to 3 groups of R^(a). Stillanother sub-embodiment of this invention is realized when the R⁶substituent on A is attached to a nitrogen atom.

Another embodiment of the present invention is realized when R² ishydrogen and R³ is (CHR^(y))_(n)pyridyl, (CHR^(y))_(n)oxidopyridyl,(CHR^(y))_(n)pyrimidinyl, (CHR^(y))_(n)triazolyl, (CHR^(y))_(n)phenyl,(CHR^(y))_(n)pyrazinyl, (CHR^(y))_(n)pyrazolyl,(CHR^(y))_(n)oxadiazolyl, (CHR^(y))_(n)thiazolyl,(CHR^(y))_(n)thiadiazolyl, (CHR^(y))_(n)indazopyridyl, C₁₋₆ alkyl, and(CHR^(y))_(n)C₃₋₆ cycloalkyl, all of which are optionally substitutedwith 1 to 3 groups of R^(a) and all other variables are as previouslydescribed. A sub-embodiment of this invention is realized when R³ is(CHR^(y))_(n)pyridyl, (CHR^(y))_(n)pyrimidinyl, (CHR^(y))_(n)triazolyl,(CHR^(y))_(n)pyrazolyl, (CHR^(y))_(n)oxadiazolyl, all of which areoptionally substituted with 1 to 3 groups of R^(a).

Another embodiment of the present invention is realized when R¹represents H, C₁₋₆ alkyl, CN, or halogen, preferably halogen selectedfrom the group consisting of fluorine and chlorine.

-   -   Another embodiment of this invention is realized by the compound        of formulas II, III, IV, V, VI, and VII:

wherein R¹, R², R^(y), R⁶ and R³, are as previously described, R^(6a) isR⁶, and Y is CH or N. A sub-embodiment of this invention is realized byanyone of the compound of formulas II, IV, V, and VI when Y is CH.Another sub-embodiment of this invention is realized by anyone of thecompound of formulas II, IV, V, and VI when Y is N. A sub-embodiment ofthis invention is realized by anyone of the compound of formulas II,III, IV, V, VI, and VII wherein R¹ is H, halogen, CN, or C₁₋₆ alkyl; R²is H; R⁶ and R^(6a) are independently selected from the group consistingof hydrogen, C₁₋₆ alkyl, (CH₂)_(n)C₃₋₁₀ cycloalkyl, (CH₂)_(n)C₆₋₁₀ aryl,(CH₂)_(n)C₅₋₁₀ heterocyclyl, said alkyl, aryl, heterocyclyl optionallysubstituted with 1 to 3 groups of R^(a); and R³ is selected from thegroup consisting of (CHR^(y))_(n)pyridyl, (CHR^(y))_(n)oxidopyridyl,(CHR^(y))_(n)pyrimidinyl, (CHR^(y))_(n)triazolyl,(CHR^(y))_(n)thiadiiazolyl, (CHR^(y))_(n)phenyl, (CHR^(y))_(n)pyrazinyl,(CHR^(y))_(n)pyrazolyl, (CHR^(y))_(n)oxadiazolyl,(CHR^(y))_(n)thiazolyl, C₁₋₆ alkyl, and (CHR^(y))_(n)C₃₋₆ cycloalkyl,all of which are optionally substituted with 1 to 3 groups of R^(a). Afurther sub-embodiment of the compounds of formulas II, III, IV, V, VI,and VII is realized when R^(6a) is selected from the group consisting of(CHR^(y))_(n)pyridyl, (CHR^(y))_(n)thiophenyl, (CHR^(y))_(n)pyrimidinyl,(CHR^(y))_(n)phenyl, (CHR^(y))_(n)C₃₋₁₀ cycloalkyl, and C₁₋₆ alkyl, allof which are optionally substituted with 1 to 3 groups of R^(a), and R³is selected from the group consisting of (CHR^(y))_(n)pyridyl,(CHR^(y))_(n)oxidopyridyl, (CHR^(y))_(n)pyrimidinyl,(CHR^(y))_(n)triazolyl, (CHR^(y))_(n)pyrazinyl, (CHR^(y))_(n)pyrazolyl,and (CHR^(y))_(n)oxadiazolyl, all of which are optionally substitutedwith 1 to 3 groups of R^(a).

Still another embodiment of this invention is realized with thecompounds of formula II and all variables are as previously described.

Another embodiment of this invention is realized with the compounds offormula III and all variables are as previously described.

Another embodiment of this invention is realized with the compounds offormula IV and all variables are as previously described.

Another embodiment of this invention is realized with the compounds offormula V and all variables are as previously described.

Another embodiment of this invention is realized with the compounds offormula VI and all variables are as previously described.

Another embodiment of this invention is realized with the compounds offormula VII and all variables are as previously described.

Examples of compounds of this invention are found in Tables 1-6:

TABLE 1

Example R³ B R⁶ MS (M + 1) 1.1

447.1111 1.2

431.1166 1.3

381.1178 1.4

432.1123 1.5

353.1188 1.6

368.1180 1.7

383.0996 1.8

397.1128 1.9

417.0982 1.10

451.0596 1.11

385.0926 1.12

399.1080 1.13

479.1171 1.14

461.1248 1.15

465.0993 1.16

481.0703 1.17

515.0955 1.18

461.1258 1.19

465.0991 1.20

481.0699 1.21

515.0960 1.22

461.1249 1.23

465.0992 1.24

481.0703 1.25

515.0956 1.26

448.1046 1.27

462.1201 1.28

483.0929 1.29

386.1086 1.30

386.1081 1.31

401.0901 1.32

386.1082 1.33

382.1326 1.34

400.1233 1.35

404.0988 1.36

386.1080 1.37

402.0783 1.38

436.1044 1.39

410.1335 1.40

411.1286 1.41

394.1385 1.42

414.0839 1.43

381.1180 1.44

395.1337 1.45

421.1244 1.46

418.1161 1.47

432.1324 1.48

420.0946 1.49

434.1090 1.50

450.1039 1.51

434.1106 1.52

450.0880 1.53

418.1144 1.54

447.1093 1.55

498.1024 1.56

449.0908 1.57

433.1255 1.58

472.0702 1.59

462.1400 1.60

448.1243 1.61

460.1243 1.62

476.1565 1.63

481.1251 1.64

481.1262 1.65

481.1270 1.66

503.1660 1.67

448.1248 1.68

464.1194 1.69

447.1044 1.70

517.1825 1.71

482.1207 1.72

462.1404 1.73

516.1985 1.74

508.0931 1.75

482.1221 1.76

482.1206 1.77

482.1225 1.78

474.1406 1.79

459.1419 1.80

502.1838 1.81

517.1482 1.82

559.1938 1.83

461.1562 1.84

462.1418 1.85

462.1420 1.86

444.1298 1.87

448.1243 1.88

495.1406 1.89

499.1171 1.90

461.1091 1.91

485.1025 1.92

468.1057 1.93

517.1494 1.94

448.1257 1.95

474.1417 1.96

478.1367 1.97

492.1522 1.98

468.1063 1.99

468.1070 1.100

526.1328 1.101

528.1125 1.102

538.1350 1.103

542.1289 1.104

556.1453 1.105

532.0987 1.106

532.0999 1.107

478.1370 1.108

492.1523 1.109

468.1070 1.110

456.3

TABLE 2

Example R³ B R⁶ MS (M + 1) 2.1

434.1091

TABLE 3

Example R³ B R⁶ MS (M + 1) 3.1

472.1551 3.2

457.1557 3.3

551.1479 3.4

536.1484 3.5

404.1526 3.6

488.1299 3.7

488.1 3.8

485.1528 3.9

503.2203 3.10

474.1690 3.11

478.1682 3.12

473.1195 3.13

473.1488 3.14

457.1425 3.15

456.1587 3.16

480.1143 3.17

549.1503 3.18

487.1356 3.19

488.1479 3.20

457.1551 3.21

488.1308 3.22

551.1454 3.23

521.2095 3.24

484.1893 3.25

533.1840 3.26

484.1889 3.27

472.1546 3.28

540.1427 3.29

500.1846 3.30

486.1689 3.31

498.1688 3.32

502.1636 3.33

486.1690 3.34

486.1688 3.35

486.1690 3.36

420.1477 3.37

420.1477 3.38

420.1475 3.39

434.1633 3.40

472.1551 3.41

489.2058 3.42

424.1530 3.43

409.1535 3.44

503.1466 3.45

488.1462 3.46

422.1991 3.47

501.1919 3.48

486.1924 3.49

426.1741 3.50

505.1668 3.51

490.1670 3.52

475.1149 3.53

460.1147 3.54

396.1227 3.55

381.1223 3.56

412.0988 3.57

380.1516 3.58

444.1443 3.59

459.1450 3.60

476.1096 3.61

461.1100 3.62

476.1287 3.63

492.1237 3.64

555.1222 3.65

461.1297 3.66

492.1063 3.67

540.1210 3.68

555.1204 3.69

493.0943 3.70

509.0895 3.71

572.0872 3.72

509.0712 3.73

458.1393 3.74

458.1386 3.75

474.1346 3.76

443.1406 3.77

522.1320 3.78

537.1324 3.79

492.0989 3.80

382.2 3.81

477.1248 3.82

493.2000 3.83

488.1489 3.84

500.1833 3.85

526.1240 3.86

473.1490 3.87

473.1484 3.88

478.1094 3.89

474.1435 3.90

474.1432 3.91

472.1532 3.92

490.1431 3.93

486.1689 3.94

486.1685 3.95

464.0931 3.96

448.1162 3.97

464.0931 3.98

478.1091 3.99

462.1435 3.100

448.1181 3.101

479.1048 3.102

465.0903 3.103

465.0897 3.104

466.0850 3.105

426.1328 3.106

454.1645 3.107

467.1628 3.108

452.1631 3.109

467.1627 3.110

546.1552 3.111

531.1562 3.112

464.1826 3.113

453.1787 3.114

468.1585 3.115

527.1515 3.116

470.1407 3.117

470.1405 3.118

473. 206 3.119

552.1128 3.120

537.1137 3.121

473.1208 3.122

H

362.1611 3.123

H

441.1535 3.124

H

426.1531 3.125

473.1476 3.126

552.1393 3.127

537.1398 3.128

456.1828 3.129

535.1756 3.130

520.1755 3.131

457.1784 3.132

536.1706 3.133

521.1707 3.134

474.1748 3.135

459.1748 3.136

553.1673 3.137

538.1677 3.138

470.2004 3.139

455.2002 3.140

549.1923 3.141

534.1929 3.142

474.1739 3.143

459.1747 3.144

553.1681 3.145

538.1677 3.146

524.1493 3.147

490.1498 3.148

487.1734 3.149

490.1682 3.150

440.0730 3.151

490.1445 3.152

475.1446 3.153

569.1381 3.154

554.1373 3.155

452.2079 3.156

437.2087 3.157

531.1997 3.158

516.2004 3.159

480.2395 3.160

465.2396 3.161

559.2322 3.162

544.2318 3.163

468.2031 3.164

453.2036 3.165

547.1959 3.166

532.1956 3.167

506.1797 3.168

491.1801 3.169

585.1723 3.170

570.1731 3.171

473.1489 3.172

458.1490 3.173

552.1420 3.174

537.1416 3.175

463.1885 3.176

527.1806 3.177

479.1823 3.178

448.1895 3.179

542.1802 3.180

440.1834 3.181

456.1780 3.182

425.1842 3.183

519.1768 3.184

504.1765 3.185

506.1141 3.186

522.1096 3.187

491.1148 3.188

585.1064 3.189

570.1067 3.190

522.0918 3.191

585.1059 3.192

444.2394 3.193

460.2343 3.194

429.2397 3.195

458.1642 3.196

443.1661 3.197

537.1592 3.198

522.1574 3.199

404.2080 3.200

389.2118 3.201

483.2006 3.202

468.2023 3.203

420.2047 3.204

420.2045 3.205

405.2036 3.206

521.4 (M + 23 (Na)) 3.207

484.1955 3.208

436.1969 3.209

436.1988 3.210

436.1988 3.211

438.1923 3.212

516.1723 3.213

472.1552 3.214

477.2029 3.215

456.1830 3.216

464.1833 3.217

526.2081 3.218

511.2086 3.219

439.1874 3.220

474.1731 3.221

454.1876 3.222

454.1870 3.223

468.2032 3.224

498.2134 3.225

498.2135 3.226

463.1879 3.227

452.2079 3.228

452.2078 3.229

495.2139 3.230

428.1713 3.231

428.1715 3.232

444.1485 3.233

442.1986 3.234

506.1794 3.235

482.1821 3.236

427.1878 3.237

537.2603 3.238

522.1745 3.239

481.2339 3.240

493.1982 3.241

506.1138 3.242

472.1534 3.243

474.1734 3.244

468.2030 3.245

469.1446 3.246

402.1928 3.247

392.1728 3.248

460.1607 3.249

447.2138 3.250

446.2297 3.251

412.1452 3.252

428.1222 3.253

428.1400 3.254

412.1450 3.255

425.1444 3.256

443.1349 3.257

428.1386 3.258

444.1350 3.259

428.1413 3.260

452.1013 3.261

452.1021 3.262

396.1490 3.263

412.1436 3.264

396.1491 3.265

397.1437 3.266

435.1368 3.267

412.1442 3.268

427.1542 3.269

414.1240 3.270

411.1599 3.271

425.1385 3.272

439.1545 3.273

452.1860 3.274

438.1705 3.275

453.1357 3.276

426.1599 3.277

426.1608 3.278

490.1529 3.279

442.1379 3.280

442.1549 3.281

442.1556 3.282

427.1555 3.283

506.1471 3.284

458.1317 3.285

466.1168 3.286

465.1321 3.287

411.1606 3.288

426.1596 3.289

425.1756 3.290

412.1448 3.291

412.1447 3.292

424.1794 3.293

424.1798 3.294

409.1789 3.295

525.3 (M + 23(Na)) 3.296

488.1728 3.297

440.1488 3.298

456.1439 3.299

406.1881 3.300

422.1839 3.301

406.1886 3.302

413.1413 3.303

413.1408 3.304

422.1947 3.305

422.1943 3.306

413.1394 3.307

470.1407 3.308

412.1441 3.309

396.1681 3.310

412.1629 3.311

443.1507 3.312

428.1390 3.313

412.1634 3.314

428.1572 3.315

422.1835 3.316

438.1791 3.317

422.1824 3.318

456.1439 3.319

472.1387 3.320

504.1675 3.321

453.0948 3.322

453.0947 3.323

450.1500

TABLE 4

Example R³ R^(6a) R⁶ MS (M + 1) 4.1

472.1532 4.2

488.1502 4.3

457.1549 4.4

551.1476

TABLE 5

Example R³ B R⁶ MS (M + 1) 5.1

H 444.1338 5.2

H 429.1336 5.3

H 365.1413 5.4

458.1491 5.5

443.1497 5.6

379.1566 5.7

364.1565 5.8

486.1798 5.9

534.1800

TABLE 6

Example R³ B R⁶ MS (M + 1) 6.1

456.1827 6.2

535.1759 6.3

520.1753 6.4

473.1794 6.5

458.1799 6.6

552.1727 6.7

537.1726 6.8

473.4990 6.9

552.1728 6.10

537.1725 6.11

471.1585 6.12

456.1587 6.13

550.1505 6.14

535.1508 6.15

375.1259 6.16

424.1526 6.17

488.1476 6.18

503.1474 6.19

423.1580 6.20

408.1578 6.21

502.1504 6.22

487.1499 6.23

472.1525 6.24

551.1444 6.25

536.1446 6.26

425.1775 6.27

504.1696 6.28

489.1697 6.29

548.1967 6.30

534.1802 6.31

519.1793 6.32

469.2036 6.33

455.1875or pharmaceutically acceptable salts and individual enantiomers anddiastereomers thereof.

When any variable (e.g. aryl, heterocycle, R¹, R⁵ etc.) occurs more thanone time in any constituent, its definition on each occurrence isindependent at every other occurrence. Also, combinations ofsubstituents/or variables are permissible only if such combinationsresult in stable compounds.

When R^(a) is —O— and attached to a carbon it is referred to as acarbonyl group and when it is attached to a nitrogen (e.g., nitrogenatom on a pyridyl group) or sulfur atom it is referred to a N-oxide andsulfoxide group, respectively.

As used herein, “alkyl” encompasses groups having the prefix “alk” suchas, for example, alkoxy, alkanoyl, alkenyl, and alkynyl and means carbonchains which may be linear or branched or combinations thereof. Examplesof alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-and tert-butyl, pentyl, hexyl, and heptyl. “Alkenyl” refers to ahydrocarbon radical straight, branched or cyclic containing from 2 to 10carbon atoms and at least one carbon to carbon double bond. Preferredalkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl.Preferably, alkenyl is C₂-C₆ alkenyl. Preferred alkynyla are C₂-C₆alkynyl. “Alkenyl,” “alkynyl” and other like terms include carbon chainscontaining at least one unsaturated C—C bond.

As used herin, “fluoroalkyl” refers to an alkyl substituent as describedherin containing at least one flurine substituent.

The compounds of this invention include N-oxides such as those disclosedin the definition of R³.

The term “cycloalkyl” refers to a saturated hydrocarbon containing onering having a specified number of carbon atoms. Examples of cycloalkylinclude cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term “C₁₋₆” includes alkyls containing 6, 5, 4, 3, 2, or 1 carbonatoms

The term “alkoxy” as used herein, alone or in combination, includes analkyl group connected to the oxy connecting atom. The term “alkoxy” alsoincludes alkyl ether groups, where the term ‘alkyl’ is defined above,and ‘ether’ means two alkyl groups with an oxygen atom between them.Examples of suitable alkoxy groups include methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, s-butoxy, t-butoxy, methoxymethane (also referredto as ‘dimethyl ether’), and methoxyethane (also referred to as ‘ethylmethyl ether’).

As used herein, “aryl” is intended to mean any stable monocyclic orbicyclic carbon ring of up to 7 members in each ring, wherein at leastone ring is aromatic. Examples of such aryl elements include phenyl,napthyl, tetrahydronapthyl, indanyl, or biphenyl.

The term heterocycle, heterocyclyl, or heterocyclic, as used herein,represents a stable 5- to 7-membered monocyclic or stable 8- to11-membered bicyclic heterocyclic ring which is either saturated orunsaturated, and which consists of carbon atoms and from one to fourheteroatoms selected from the group consisting of N, O, and S, andincluding any bicyclic group in which any of the above-definedheterocyclic rings is fused to a benzene ring. The heterocyclic ring maybe attached at any heteroatom or carbon atom which results in thecreation of a stable structure. The term heterocycle or heterocyclicincludes heteroaryl moieties. Examples of such heterocyclic elementsinclude, but are not limited to, azepinyl, benzimidazolyl,benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl,benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl,cinnolinyl, dihydrobenzofuryl, dihydrobenzothienyl,dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone,1,3-dioxolanyl, furyl, imidazolidinyl, imidazolinyl, imidazolyl,indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl,isothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholinyl,naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl,pyrazinyl, pyrazolidinyl, pyrazolyl, pyridazinyl, pyrimidinyl,pyrrolidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl,tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,thiamorpholinyl, thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl,thienofuryl, thienothienyl, thienyl and triazolyl.

In certain embodiments, the heterocyclic group is a heteroaryl group. Asused herein, the term “heteroaryl” refers to groups having 5 to 14 ringatoms, preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or 14 πelectrons shared in a cyclic array; and having, in addition to carbonatoms, between one and about three heteroatoms selected from the groupconsisting of N, 0, and S. heteroaryl groups include, withoutlimitation, thienyl, benzothienyl, furyl, benzofuryl, dibenzofuryl,pyrrolyl, imidazolyl, pyrazoiyl, pyridyl, pyrazinyl, pyrimidinyl,indolyl, quinolyl, isoquinolyl, quinoxalinyl, tetrazolyl, oxazolyl,thiazolyl, and isoxazolyl.

In certain other embodiments, the heterocyclic group is fused to an arylor heteroaryl group. Examples of such fused heterocycles include,without limitation, tetrahydroquinolinyl and dihydrobenzofuranyl.

The term “heteroaryl”, as used herein except where noted, represents astable 5- to 7-membered monocyclic- or stable 9- to 10-membered fusedbicyclic heterocyclic ring system which contains an aromatic ring, anyring of which may be saturated, such as piperidinyl, partiallysaturated, or unsaturated, such as pyridinyl, and which consists ofcarbon atoms and from one to four heteroatoms selected from the groupconsisting of N, O and S, and wherein the nitrogen and sulfurheteroatoms may optionally be oxidized, and the nitrogen heteroatom mayoptionally be quaternized, and including any bicyclic group in which anyof the above-defined heterocyclic rings is fused to a benzene ring. Theheterocyclic ring may be attached at any heteroatom or carbon atom whichresults in the creation of a stable structure. Examples of suchheteroaryl groups include, but are not limited to, benzimidazole,benzisothiazole, benzisoxazole, benzofuran, benzothiazole,benzothiophene, benzotriazole, benzoxazole, carboline, cinnoline, furan,furazan, imidazole, indazole, indole, indolizine, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, phthalazine,pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine,pyrimidine, pyrrole, quinazoline, quinoline, quinoxaline, tetrazole,thiadiazole, thiazole, thiophene, triazine, triazole, and N-oxidesthereof.

Examples of heterocycloalkyls include azetidinyl, pyrrolidinyl,piperidinyl, piperazinyl, morpholinyl, tetrahydrofuranyl, imidazolinyl,pyrrolidin-2-one, piperidin-2-one, and thiomorpholinyl.

The term “heteroatom” means O, S or N, selected on an independent basis.

A moiety that is substituted is one in which one or more hydrogens havebeen independently replaced with another chemical substituent. As anon-limiting example, substituted phenyls include 2-fluorophenyl,3,4-dichlorophenyl, 3-chloro-4-fluoro-phenyl, 2,4-fluor-3-propylphenyl.As another non-limiting example, substituted n-octyls include 2,4dimethyl-5-ethyl-octyl and 3-cyclopentyloctyl. Included within thisdefinition are methylenes (—CH₂—) substituted with oxygen to formcarbonyl (—CO—).

Unless otherwise stated, as employed herein, when a moiety (e.g.,cycloalkyl, hydrocarbyl, aryl, alkyl, heteroaryl, heterocyclic, urea,etc.) is described as “optionally substituted” it is meant that thegroup optionally has from one to four, preferably from one to three,more preferably one or two, non-hydrogen substituents. Suitablesubstituents include, without limitation, halo, hydroxy, oxo (e.g., anannular —CH— substituted with oxo is —C(O)—), nitro, halohydrocarbyl,hydrocarbyl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino,alkylcarbamoyl, arylcarbamoyl, aminoalkyl, acyl, carboxy, hydroxyalkyl,alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido,aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups.Preferred substituents, which are themselves not further substituted(unless expressly stated otherwise) are:

-   -   (a) halo, cyano, oxo, carboxy, formyl, nitro, amino, amidino,        guanidino, and    -   (b) C₁-C₆ alkyl or alkenyl or arylalkyl imino, carbamoyl, azido,        carboxamido, mercapto, hydroxy, hydroxyalkyl, alkylaryl,        arylalkyl, C₁-C₈ alkyl, SO₂CF₃, CF₃, SO₂Me, C₁-C₈ alkenyl, C₁-C₈        alkoxy, C₁-C₈ alkoxycarbonyl, aryloxycarbonyl, C₂-C₈ acyl, C₂-C₈        acylamino, C₁-C₈ alkylthio, arylalkylthio, arylthio,        C₁-C₈alkylsulfinyl, arylalkylsulfnyl arylsulfnyl, C₁-C₈        alkylsulfonyl, arylalkylsulfonyl, arylsulfonyl, C₀-C₆        N-alkylcarbamoyl, C₂-C₁₅ N,N dialkylcarbamoyl, C₃-C₇ cycloalkyl,        aroyl, aryloxy, arylalkyl ether, aryl, aryl fused to a        cycloalkyl or heterocycle or another aryl ring, C₃-C₇        heterocycle, or any of these rings fused or spiro-fused to a        cycloalkyl, heterocyclyl, or aryl, wherein each of the foregoing        is further optionally substituted with one more moieties listed        in (a), above.

“Halogen” refers to fluorine, chlorine, bromine and iodine.

The term “mammal” “mammalian” or “mammals” includes humans, as well asanimals, such as dogs, cats, horses, pigs and cattle.

All patents, patent applications and publications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretyand are deemed representative of the prevailing state of the art.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise. Thus, for example, reference to “a primer”includes two or more such primers, reference to “an amino acid” includesmore than one such amino acid, and the like.

The phrases “effective amount” or “therapeutically effective amount”mean a concentration of P2X receptor complex modulator sufficient toinhibit or enhance the effect of the P2X receptor complex.

“Pain” means the more or less localized sensation of discomfort,distress, or agony, resulting from the stimulation of specialized nerveendings. There are many types of pain, including, but not limited to,lightning pains, phantom pains, shooting pains, acute pain, inflammatorypain, neuropathic pain, complex regional pain, neuralgia, neuropathy,tissue injury pain, and the like (Dorland's Illustrated MedicalDictionary, 28th Edition, W. B. Saunders Company, Philadelphia, Pa.).The goal of treatment of pain is to reduce the degree or severity ofpain perceived by a treatment subject.

Compounds described herein may contain one or more double bonds and maythus give rise to cis/trans isomers as well as other conformationalisomers. The present invention includes all such possible isomers aswell as mixtures of such isomers unless specifically stated otherwise.

The compounds of the present invention may contain one or moreasymmetric centers and may thus occur as racemates, racemic mixtures,single enantiomers, diastereomeric mixtures, and individualdiastereomers.

In the compounds of generic Formula I, the atoms may exhibit theirnatural isotopic abundances, or one or more of the atoms may beartificially enriched in a particular isotope having the same atomicnumber, but an atomic mass or mass number different from the atomic massor mass number predominantly found in nature. The present invention ismeant to include all suitable isotopic variations of the compounds ofgeneric Formula I. For example, different isotopic forms of hydrogen (H)include protium (¹H) and deuterium (²H). Protium is the predominanthydrogen isotope found in nature. Enriching for deuterium may affordcertain therapeutic advantages, such as increasing in vivo half-life orreducing dosage requirements, or may provide a compound useful as astandard for characterization of biological samples.Isotopically-enriched compounds within generic Formula I can be preparedwithout undue experimentation by conventional techniques well known tothose skilled in the art or by processes analogous to those described inthe Schemes and Examples herein using appropriate isotopically-enrichedreagents and/or intermediates.

It will be understood that, as used herein, references to the compoundsof structural formula I are meant to also include the pharmaceuticallyacceptable salts, and also salts that are not pharmaceuticallyacceptable when they are used as precursors to the free compounds or inother synthetic manipulations.

The compounds of the present invention may be administered in the formof a pharmaceutically acceptable salt. The term “pharmaceuticallyacceptable salts” refers to salts prepared from pharmaceuticallyacceptable non-toxic bases or acids. When the compound of the presentinvention is acidic, its corresponding salt can be conveniently preparedfrom pharmaceutically acceptable non-toxic bases, including inorganicbases and organic bases. Salts derived from such inorganic bases includealuminum, ammonium, calcium, copper (ic and ous), ferric, ferrous,lithium, magnesium, manganese (ic and ous), potassium, sodium, zinc andthe like salts. Salts derived from pharmaceutically acceptable organicnon-toxic bases include salts of primary, secondary, and tertiaryamines, as well as cyclic amines and substituted amines such asnaturally occurring and synthesized substituted amines. Otherpharmaceutically acceptable organic non-toxic bases from which salts canbe formed include ion exchange resins such as, for example, arginine,betaine, caffeine, choline, N, dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, and tromethamine.

When the compound of the present invention is basic, its correspondingsalt can be conveniently prepared from pharmaceutically acceptablenon-toxic acids, including inorganic and organic acids. Such acidsinclude, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic,citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic,hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric,succinic, sulfuric, tartaric, p-toluenesulfonic acid and the like.

The pharmaceutical compositions of the present invention comprisecompounds of the invention (or pharmaceutically acceptable saltsthereof) as an active ingredient, a pharmaceutically acceptable carrier,and optionally one or more additional therapeutic agents or adjuvants.Such additional therapeutic agents can include, for example, i) opiateagonists or antagonists, ii) calcium channel antagonists, iii) 5HTreceptor agonists or antagonists, iv) sodium channel antagonists, v)NMDA receptor agonists or antagonists, yl) COX-2 selective inhibitors,vii) NK1 antagonists, viii) non-steroidal anti-inflammatory drugs(“NSAID”), ix) selective serotonin reuptake inhibitors (“SSRI”) and/orselective serotonin and norepinephrine reuptake inhibitors (“SSNRI”), x)tricyclic antidepressant drugs, xi) norepinephrine modulators, xii)lithium, xiii) valproate, xiv) neurontin (gabapentin), xv) pregabalin,and xvi) sodium channel blockers. The instant compositions includecompositions suitable for oral, rectal, topical, and parenteral(including subcutaneous, intramuscular, and intravenous) administration,although the most suitable route in any given case will depend on theparticular host, and nature and severity of the conditions for which theactive ingredient is being administered. The pharmaceutical compositionsmay be conveniently presented in unit dosage form and prepared by any ofthe methods well known in the art of pharmacy.

The present compounds and compositions are useful for the treatment ofchronic, visceral, inflammatory and neuropathic pain syndromes. They areuseful for the treatment of pain resulting from traumatic nerve injury,nerve compression or entrapment, postherpetic neuralgia, trigeminalneuralgia, small fiber neuropathy, and diabetic neuropathy. The presentcompounds and compositions are also useful for the treatment of chroniclower back pain, phantom limb pain, chronic pelvic pain, neuroma pain,complex regional pain syndrome, chronic arthritic pain and relatedneuralgias, and pain associated with cancer, chemotherapy, HIV and HIVtreatment-induced neuropathy. Compounds of this invention may also beutilized as local anesthetics. Compounds of this invention are usefulfor the treatment of irritable bowel syndrome and related disorders, aswell as Crohn's disease.

The instant compounds have clinical uses for the treatment of epilepsyand partial and generalized tonic seizures. They are also useful forneuroprotection under ischaemic conditions caused by stroke or neuraltrauma and for treating multiple sclerosis. The present compounds areuseful for the treatment of tachy-arrhythmias. Additionally, the instantcompounds are useful for the treatment of neuropsychiatric disorders,including mood disorders, such as depression or more particularlydepressive disorders, for example, single episodic or recurrent majordepressive disorders and dysthymic disorders, or bipolar disorders, forexample, bipolar I disorder, bipolar II disorder and cyclothymicdisorder; anxiety disorders, such as panic disorder with or withoutagoraphobia, agoraphobia without history of panic disorder, specificphobias, for example, specific animal phobias, social phobias,obsessive-compulsive disorder, stress disorders including post-traumaticstress disorder and acute stress disorder, and generalised anxietydisorders. Thus, another aspect of this invention is the use of thecompounds of formula I in the manufacture of a medicament to treat painand other diseases associated with pain.

In addition to primates, such as humans, a variety of other mammals canbe treated according to the method of the present invention. Forinstance, mammals including, but not limited to, cows, sheep, goats,horses, dogs, cats guinea pigs, or other bovine, ovine, equine, canine,feline, rodent such as mouse, species can be treated. However, themethod can also be practiced in other species, such as avian species(e.g., chickens).

It will be appreciated that for the treatment of depression or anxiety,a compound of the present invention may be used in conjunction withother anti-depressant or anti-anxiety agents, such as norepinephrinereuptake inhibitors, selective serotonin reuptake inhibitors (SSRIs),monoamine oxidase inhibitors (MAOIs), reversible inhibitors of monoamineoxidase (RIMAs), serotonin and noradrenaline reuptake inhibitors(SNRIs), α-adrenoreceptor antagonists, atypical anti-depressants,benzodiazepines, 5-HT_(1A) agonists or antagonists, especially 5-HT_(1A)partial agonists, neurokinin-1 receptor antagonists, corticotropinreleasing factor (CRF) antagonists, and pharmaceutically acceptablesalts thereof.

Further, it is understood that compounds of this invention can beadministered at prophylactically effective dosage levels to prevent theabove-recited conditions and disorders, as well as to prevent otherconditions and disorders associated with calcium channel activity.

Creams, ointments, jellies, solutions, or suspensions containing theinstant compounds can be employed for topical use. Mouth washes andgargles are included within the scope of topical use for the purposes ofthis invention.

Dosage levels from about 0.01 mg/kg to about 140 mg/kg of body weightper day are useful in the treatment of inflammatory and neuropathicpain, or alternatively about 0.5 mg to about 7 g per patient per day.For example, inflammatory pain may be effectively treated by theadministration of from about 0.01 mg to about 75 mg of the compound perkilogram of body weight per day, or alternatively about 0.5 mg to about3.5 g per patient per day. Neuropathic pain may be effectively treatedby the administration of from about 0.01 mg to about 125 mg of thecompound per kilogram of body weight per day, or alternatively about 0.5mg to about 5.5 g per patient per day.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, aformulation intended for the oral administration to humans mayconveniently contain from about 0.5 mg to about 5 g of active agent,compounded with an appropriate and convenient amount of carrier materialwhich may ary from about 5 to about 95 percent of the total composition.Unit dosage forms will generally contain between from about 1 mg toabout 1000 mg of the active ingredient, typically 25 mg, 50 mg, 100 mg,200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg or 1000 mg.

It is understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors. Suchpatient-related factors include the age, body weight, general health,sex, and diet of the patient. Other factors include the time and routeof administration, rate of excretion, drug combination, and the severityof the particular disease undergoing therapy.

In practice, the compounds of the invention, or pharmaceuticallyacceptable salts thereof, can be combined as the active ingredient inintimate admixture with a pharmaceutical carrier according toconventional pharmaceutical compounding techniques. The carrier may takea wide variety of forms depending on the form of preparation desired foradministration, e.g., oral or parenteral (including intravenous). Thus,the pharmaceutical compositions of the present invention can bepresented as discrete units suitable for oral administration such ascapsules, cachets or tablets each containing a predetermined amount ofthe active ingredient. Further, the compositions can be presented as apowder, as granules, as a solution, as a suspension in an aqueousliquid, as a non-aqueous liquid, as an oil-in-water emulsion or as awater-in-oil liquid emulsion. In addition to the common dosage forms setout above, the compounds of the invention, or pharmaceuticallyacceptable salts thereof, may also be administered by controlled releasemeans and/or delivery devices. The compositions may be prepared by anyof the methods of pharmacy. In general, such methods include a step ofbringing into association the active ingredient with the carrier thatconstitutes one or more necessary ingredients. In general, thecompositions are prepared by uniformly and intimately admixing theactive ingredient with liquid carriers or finely divided solid carriersor both. The product can then be conveniently shaped into the desiredpresentation.

Thus, the pharmaceutical compositions of this invention may include apharmaceutically acceptable carrier and a compound or a pharmaceuticallyacceptable salt. The compounds of the invention, or pharmaceuticallyacceptable salts thereof, can also be included in pharmaceuticalcompositions in combination with one or more therapeutically activecompounds.

The pharmaceutical carrier employed can be, for example, a solid,liquid, or gas. Examples of solid carriers include lactose, terra alba,sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, andstearic acid. Examples of liquid carriers are sugar syrup, peanut oil,olive oil, and water. Examples of gaseous carriers include carbondioxide and nitrogen. As described previously, in preparing thecompositions for oral dosage form, any of the usual pharmaceutical mediacan be employed. For example, in the case of oral liquid preparationssuch as suspensions, elixirs and solutions, water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents and the likemay be used; or in the case of oral solid preparations such as powders,capsules and tablets, carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents, and the like may be included. Because oftheir ease of administration, tablets and capsules represent the mostadvantageous oral dosage unit form in which solid pharmaceuticalcarriers are employed. If desired, tablets may be coated by standardaqueous or nonaqueous techniques. In addition to the common dosage formsset out above, controlled release means and/or delivery devices may alsobe used in administering the instant compounds and compositions.

In preparing the compositions for oral dosage form, any convenientpharmaceutical media may be employed. For example, water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents and the likemay be used to form oral liquid preparations such as suspensions,elixirs and solutions; while carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents can be used to form oral solidpreparations such as powders, capsules and tablets. Because of theirease of administration, tablets and capsules are advantageous oraldosage units whereby solid pharmaceutical carriers are employed.Optionally, tablets may be coated by standard aqueous or nonaqueoustechniques

A tablet containing the composition of this invention may be prepared bycompression or molding, optionally with one or more accessoryingredients or adjuvants. Compressed tablets may be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets may be made by molding in a suitable machine, a mixtureof the powdered compound moistened with an inert liquid diluent. Eachtablet advantageously contains from about 0.1 mg to about 500 mg of theactive ingredient and each cachet or capsule advantageously containingfrom about 0.1 mg to about 500 mg of the active ingredient. Thus, atablet, cachet, or capsule conveniently contains 0.1 mg, 1 mg, 5 mg, 25mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, or 500 mg of the activeingredient taken one or two tablets, cachets, or capsules, once, twice,or three times daily.

Pharmaceutical compositions of the present invention suitable forparenteral administration may be prepared as solutions or suspensions ofthe active compounds in water. A suitable surfactant can be includedsuch as, for example, hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofin oils. Further, a preservative can be included to prevent thedetrimental growth of microorganisms.

Pharmaceutical compositions of the present invention suitable forinjectable use include sterile aqueous solutions or dispersions.Furthermore, the compositions can be in the form of sterile powders forthe extemporaneous preparation of such sterile injectable solutions ordispersions. In all cases, the final injectable form must be sterile andmust be effectively fluid for easy syringability. The pharmaceuticalcompositions must be stable under the conditions of manufacture andstorage, and thus should be preserved against the contaminating actionof microorganisms such as bacteria and fungi. The carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol),vegetable oils, and suitable mixtures thereof.

Pharmaceutical compositions of the present invention can be in a formsuitable for topical use such as, for example, an aerosol, cream,ointment, lotion, and dusting powder. Further, the compositions can bein a form suitable for use in transdermal devices. These formulationsmay be prepared, utilizing a compound represented of the invention, orpharmaceutically acceptable salts thereof, via conventional processingmethods. As an example, a cream or ointment is prepared by mixinghydrophilic material and water, together with about 5 wt % to about 10wt % of the compound, to produce a cream or ointment having a desiredconsistency.

Pharmaceutical compositions of this invention can be in a form suitablefor rectal administration wherein the carrier is a solid, such as, forexample, where the mixture forms unit dose suppositories. Suitablecarriers include cocoa butter and other materials commonly used in theart. The suppositories may be conveniently formed by first admixing thecomposition with the softened or melted carriers) followed by chillingand shaping in moulds.

In addition to the aforementioned carrier ingredients, thepharmaceutical formulations described above may include, as appropriate,one or more additional carrier ingredients such as diluents, buffers,flavoring agents, binders, surface-active agents, thickeners,lubricants, and preservatives (including anti-oxidants). Furthermore,other adjuvants can be included to render the formulation isotonic withthe blood of the intended recipient. Compositions containing a compoundof the invention, or pharmaceutically acceptable salts thereof, can alsobe prepared in powder or liquid concentrate form.

Further, as described above, the instant compounds can be utilized incombination with one or more therapeutically active compounds. Inparticular, the inventive compounds can be advantageously used incombination with i) opiate agonists or antagonists, ii) other calciumchannel antagonists, iii) 5HT receptor agonists or antagonists,including 5-HT_(1A) agonists or antagonists, and 5-HT_(1A) partialagonists, iv) sodium channel antagonists, v) N-methyl-D-aspartate (NMDA)receptor agonists or antagonists, vi) COX-2 selective inhibitors, vii)neurokinin receptor 1 (NK1) antagonists, viii) non-steroidalanti-inflammatory drugs (NSAID), ix) selective serotonin reuptakeinhibitors (SSRI) and/or selective serotonin and norepinephrine reuptakeinhibitors (SSNRI), x) tricyclic antidepressant drugs, xi)norepinephrine modulators, xii) lithium, xiii) valproate, xiv)norepinephrine reuptake inhibitors, xv) monoamine oxidase inhibitors(MAOIs), xvi) reversible inhibitors of monoamine oxidase (RIMAs), xvi)alpha-adrenoreceptor antagonists, xviii) atypical anti-depressants, xix)benzodiazepines, xx) corticotropin releasing factor (CRF) antagonists,xxi) neurontin (gabapentin) and xxii) pregabalin.

The abbreviations used herein have the following meanings (abbreviationsnot shown here have their meanings as commonly used unless specificallystated otherwise): Ac (acetyl), Bn (benzyl), Boc (tertiary-butoxycarbonyl), Bop reagent(benzotriazol-1-yloxy)tris(dimethylamino)phosonium hexafluorophosphate,CAMP (cyclic adenosine-3′,5′-monophosphate), DAST ((diethylamino)sulfurtrifluoride), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), DIBAL(diisobutylaluminum hydride), DIEA (diisopropylethyl amine), DMAP(4-(dimethylamino)pyridine), DMF (N,N-dimethylformamide), DPPF(1,1′-bisdiphenylphosphino ferrocene), EDC(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), Et₃N(triethylamine), GST (glutathione transferase), HOBt(1-hydroxybenzotriazole), LAH (lithium aluminum hydride), Ms(methanesulfonyl; mesyl; or SO₂Me), MsO (methanesulfonate or mesylate),MCPBA (meta-chloro perbenzoic acid), NaHMDS (sodiumhexamethyldisilazane), NBS (N-bromosuccinimide),NCS(N-chlorosuccinimide), NSAID (non-steroidal anti-inflammatory drug),PDE (Phosphodiesterase), Ph (Phenyl), r.t. or RT (room temperature), Rac(Racemic), SAM (aminosulfonyl; sulfonamide or SO₂NH₂), SPA(scintillation proximity assay), Th (2- or 3-thienyl), TEA(trifluoroacetic acid), THE (Tetrahydrofuran), Thi (Thiophenediyl), TLC(thin layer chromatography), TMEDA(N,N,N′,N′-tetramethylethylenediamine), TMSI (trimethylsilyl iodide), Tror trityl (N-triphenylmethyl), C₃H₅ (Allyl), Me (methyl), Et (ethyl),n-Pr (normal propyl), i-Pr (isopropyl), n-Bu (normal butyl), i-Butyl(isobutyl), s-Bu (secondary butyl), t-Bu (tertiary butyl), c-Pr(cyclopropyl), c-Bu (cyclobutyl), c-Pen (cyclopentyl), c-Hex(cyclohexyl).

The present compounds can be prepared according to the proceduresprovided in the Examples. The following Examples further describe, butdo not limit, the scope of the invention.

Unless specifically stated otherwise, the experimental procedures wereperformed under the following conditions: All operations were carriedout at room or ambient temperature; that is, at a temperature in therange of 18-25° C. Inert gas protection was used when reagents orintermediates were air and moisture sensitive. Evaporation of solventwas carried out using a rotary evaporator under reduced pressure(600-4000 pascals: 4.5-30 mm Hg) with a bath temperature of up to 60° C.The course of reactions was followed by thin layer chromatography (TLC)or by high-pressure liquid chromatography-mass spectrometry (HPLC-MS),and reaction times are given for illustration only. The structure andpurity of all final products were assured by at least one of thefollowing techniques: TLC, mass spectrometry, nuclear magnetic resonance(NMR) spectrometry or microanalytical data. When given, yields are forillustration only. When given, NMR data is in the form of delta (8)values for major diagnostic protons, given in parts per million (ppm)relative to tetramethylsilane (TMS) as internal standard, determined at300 MHz, 400 MHz or 500 MHz using the indicated solvent. Conventionalabbreviations used for signal shape are: s. singlet; d. doublet; t.triplet; in. multiplet; br. Broad; etc. In addition, “Ar” signifies anaromatic signal. Chemical symbols have their usual meanings; thefollowing abbreviations are used: v (volume), w (weight), b.p. (boilingpoint), m.p. (melting point), L (liter(s)), mL (milliliters), g(gram(s)), mg (milligrams(s)), mol (moles), mmol (millimoles), eq(equivalent(s)).

The procedures described herein for synthesizing the compounds mayinclude one or more steps of protecting group manipulations and ofpurification, such as, re-crystallization, distillation, columnchromatography, flash chromatography, thin-layer chromatography (TLC),radial chromatography and high-pressure chromatography (HPLC). Theproducts can be characterized using various techniques well known in thechemical arts, including proton and carbon-13 nuclear magnetic resonance(¹H and ¹³C NMR), infrared and ultraviolet spectroscopy (IR and UV),X-ray crystallography, elemental analysis and HPLC and mass spectrometry(HPLC-MS). Methods of protecting group manipulation, purification,structure identification and quantification are well known to oneskilled in the art of chemical synthesis.

Appropriate solvents are those which will at least partially dissolveone or all of the reactants and will not adversely interact with eitherthe reactants or the product. Suitable solvents are aromatichydrocarbons (e.g, toluene, xylenes), halogenated solvents (e.g,methylene chloride, chloroform, carbontetrachloride, chlorobenzenes),ethers (e.g, diethyl ether, diisopropylether, tert-butyl methyl ether,diglyme, tetrahydrofuran, dioxane, anisole), nitriles (e.g,acetonitrile, propionitrile), ketones (e.g, 2-butanone, dithyl ketone,tert-butyl methyl ketone), alcohols (e.g, methanol, ethanol, n-propanol,iso-propanol, n-butanol, t-butanol), N,N-dimethyl formamide (DMF),dimethylsulfoxide (DMSO) and water. Mixtures of two or more solvents canalso be used. Suitable bases are, generally, alkali metal hydroxides,alkaline earth metal hydroxides such as lithium hydroxide, sodiumhydroxide, potassium hydroxide, barium hydroxide, and calcium hydroxide;alkali metal hydrides and alkaline earth metal hydrides such as lithiumhydride, sodium hydride, potassium hydride and calcium hydride; alkalimetal amides such as lithium amide, sodium amide and potassium amide;alkali metal carbonates and alkaline earth metal carbonates such aslithium carbonate, sodium carbonate, cesium carbonate, sodium hydrogencarbonate, and cesium hydrogen carbonate; alkali metal alkoxides andalkaline earth metal alkoxides such as sodium methoxide, sodiumethoxide, potassium tert-butoxide and magnesium ethoxide; alkali metalalkyls such as methyllithium, n-butyllithium, sec-butyllithium,t-bultyllithium, phenyllithium, alkyl magnaesium halides, organic basessuch as trimethylamine, triethylamine, triisopropylamine,N,N-diisopropylethyl amine, piperidine, N-methyl piperidine, morpholine,N-methyl morpholine, pyridine, collidines, lutidines, and4-dimethylaminopyridine; and bicyclic amines such as DBU and DABCO.

It is understood that the functional groups present in compoundsdescribed in the examples below can be further manipulated, whenappropriate, using the standard functional group transformationtechniques available to those skilled in the art, to provide desiredcompounds described in this invention.

It is also understood that compounds of this invention contain one ormore stereocenters that may be prepared as single enantiomers ordiastereomers, or as mixtures containing two or more enantiomers ordiastereomers in any proportion.

Other variations or modifications, which will be obvious to thoseskilled in the art, are within the scope and teachings of thisinvention. This invention is not to be limited except as set forth inthe following claims.

Several methods for preparing the compounds of this invention areillustrated in the following Schemes and Examples. Starting materialsare made according to procedures known in the art or as illustratedherein.

Reaction Schemes

The compounds of the present invention can be prepared readily accordingto the following Schemes and specific examples, or modificationsthereof, using readily available starting materials, reagents andconventional synthesis procedures. In these reactions, it is alsopossible to make use of variants which are themselves known to those ofordinary skill in this art but are not mentioned in greater detail. Thegeneral procedures for making the compounds claimed in this inventioncan be readily understood and appreciated by one skilled in the art fromviewing the following Schemes.

Amine intermediates of type 1.5 can be prepared from one of severalintermediates as shown in Scheme 1. This method utilizesdiastereoselective Ellman sulfinimine addition chemistry to generate apair of diastereomeric sulfinamides. The diastereomers are separated bysilica chromatography prior to HCl deproteetion to give 1.5. Dependingon the substrate either the R or S Ellman reagent is utilized to favorthe desired alpha methyl amino compound with the preferred stereoconfiguration shown.

Bicyclic thienopyrazole compounds of type 2.5 can be prepared asoutlined in Scheme 2. 3,4,5-Tribromo-1H-pyrazole 2.1 is converted to thealdehyde followed by alkylation with alkyl halides to give 2.2. Aldehyde2.2 undergoes ring closure to give 23 (WO 2006092510). Ester hydrolysisfollowed by EDC coupling affords 2.4, which undergoes Suzuki coupling togive final compounds of type 2.5.

Examples of type 3.5 and 3.6 can be prepared as outlined in scheme 3.3,4,5-Tribromo-1H-pyrazole 3.1 is converted to the aldehyde followed byalkylation with para-methoxybenzyl bromide to give 3.2. Aldehyde 3.2undergoes ring closure to give 3.3 (WO 2006092510). Thienopyrazole 3.3is arylated with 2,4-difluorophenylboronic acid to afford 3.4.

Removal of the para-methoxybenzyl group with TA is followed by esterhydrolysis, EDC coupling, and alkylation with alkyl halides or epoxidesor acylation with aryl halides to give final compounds of type 15 andits regioisomer, 3.6. For some compounds, the order of the final threesteps can be rearranged such that alkylation or arylation take placefirst, followed by ester hydrolysis and amide coupling.

Examples of type 4.4 can be prepared as outlined in scheme 4. Indazole4.1 undergoes alkylation with a substituted aryl bromide in the presenceof CuI (method A) or alkylation with a substituted alkyl iodide (methodB) to form intermediates of type 4.2. Bromination using NBS followed bySuzuki coupling gives 4.3. Ester hydrolysis followed by EDC couplingaffords final compounds of type 4.4.

Examples of type 5.4 can be prepared as outlined in scheme 5. Indazole5.1 is brominated with Br₂ followed by arylation with a substituted arylor heteroaryl bromide to form intermediates of type 5.2. Suzuki couplingwith aryl or alkenyl boronates gives compounds of type 5.3. An oxidationto further functionalize R^(6a) is followed by ester hydrolysis and EDCcoupling to afford compounds of type 5.4. Compounds of sub-embodimenttype VII can be prepared analogously by these methods by simply startingwith 5-(1H)indazole carboxylic acid methyl ester instead of6-(1H)indazole carboxylic acid methyl ester.

Examples of type 6.3 can be prepared as outlined in scheme 6. Bromide6.1 undergoes Suzuki coupling to give 6.2. Ester hydrolysis followed byEDC coupling affords final compounds of type 6.3.

Examples of type 7.4 can be prepared as outlined in scheme 7. Indole 7.1undergoes alkylation with a substituted aryl bromide in the presence ofCuI to form intermediates of type 7.2. Bromination using copper(II)bromide followed by Suzuki coupling gives 7.3. Ester hydrolysis followedby EDC coupling affords final compounds of type 7.4.

INTERMEDIATES AND EXAMPLES

The following examples are provided so that the invention might be morefully understood. These examples are illustrative only and should not beconstrued as limiting the invention in any way.

(1S)-1-(4H-1,2,4-Triazol-3-yl)ethanamine Step A:Benzyl[(1S)-2-amino-1-methyl-2-thioxoethyl]carbamate

To a solution of [(1S)-2-amino-1-methyl-2-oxoethyl]carbamate (15.0 g,67.5 mmol) in dichloromethane (337 mL) was added2,4-bis-(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane 2,4-disulfide(15.01 g, 37.1 mmol) and the mixture was heated to 55° C. After 1.5 h,the reaction was allowed to cool to ambient temperature and concentratedunder reduced pressure. Recrystallization from dichloromethane gave thetitle compound (13.4 g). MS 239.1 (M+1).

Step B: Benzyl[(1S)-1-(4H-1,2,4-triazol-3-yl)ethyl]carbamate

To a solution of benzyl[(1S)-2-amino-1-methyl-2-thioxoethyl]carbamate(13.4 g, 56.2 mmol) in ethanol (1.125 L) was added formic acid hydrazide(20.26 g, 337 mmol) and mercury(II) chloride (19.85 g, 73.1 mmol). After1 h the reaction was filtered and concentrated. Saturated aqueous sodiumcarbonate and ethyl acetate were added. The organic layer was isolatedand the aqueous layer was extracted with ethyl acetate (2×). Thecombined organic extracts were washed with brine, dried over magnesiumsulfate, filtered, and concentrated. A solution of the resulting residuein ethanol (1.125 L) was heated to 80° C. After 16 h, the reaction wasconcentrated. Purification by silica gel chromatography (100%dichloromethane→90% dichloromethane/methanol with 1% ammonium hydroxide)gave the title compound (8.7 g). MS 247.1 (M+1).

Step C: (1S)-1-(4H-1,2,4-Triazol-3-yl)ethanamine

To a solution of benzyl[(1S)-1-(4H-1,2,4-triazol-3-yl)ethyl]carbamate(8.6 g, 34.9 mmol) in ethanol (140 mL) was added 4 M hydrochloric acidin 1,4-dioxane (43.7 mL, 175 mmol) and 10% palladium on carbon (1.858 g,1.746 mmol) and the mixture was pressurized to 47 psi under hydrogen.After 4 h, the reaction was depressurized and filtered. Concentrationgave the title compound as a hydrochloride salt (6.6 g). MS 113.0 (M+1).¹H NMR (500 MHz, CD₃OD): δ 8.82 (s, 1H); 4.67 (q, J=6.9 Hz, 1H); 1.70(dd, J=6.9, 1.0 Hz, 3H).

(1R)-1-[6-(Trifluoromethyl)pyridin-3-yl]etanamine Step A:2-methyl-N-{(1E)-[6-(trifluoromethyl)-3-pyridinyl]methylene}-2-propanesulfinamide

To a solution of 6-(trifluoromethyl)nicotinaldehyde (45.0 g, 257 mmol)in dichloroethane (640 mL) were added(S)-(−)-2-methyl-2-propanesulfinamide (34.3 g, 283 mmol) and anhydrouscopper(II) sulfate (82 g, 514 mmol). The mixture was stirred at 50° C.After 48 h, the mixture cooled to ambient temperature. The reactionmixture was filtered through Celite. The filtered cake was washed withdichloromethane and the filtrate was concentrated to give the titlecompound (76.8 g). MS 223.1 (M-tert-butyl+1)

Step. B:2-Methyl-N-{(1R)-1-[6-(trifluoromethyl)-3-pyridinyl]ethyl}-2-propanesulfinamide

To a solution of2-methyl-N-{(1E)-[6-(trifluoromethyl)-3-pyridinyl]methylene}-2-propanesulfinamide(76.8 g, 276 mmol) in dichloromethane (920 mL) at −45° C. was addedmethylmagnesium bromide (3.0 M in tetrahydrofuran; 184 mL, 552 mmol).The mixture was stirred at −45° C. for 4 h. The reaction mixture waswarmed to −20° C. Additional methylmagnesium bromide (3.0 M intetrahydrofuran; 276 mL, 828 mmol) was added at −20° C. The reactionmixture was warmed to 0° C. and was quenched with saturated aqueousammonium chloride (300 mL). The mixture was allowed to warm to ambienttemperature. The organic layer was separated and the aqueous layer wasextracted with dichloromethane (3×). The combined organic extracts werewashed with saturated aqueous sodium chloride, dried over magnesiumsulfate, filtered and concentrated under reduced pressure. Theconcentrate was recrystallized using ethyl alcohol (500 mL). The titlecompound was filtered and dried under reduced pressure (41.6 g). MS295.0 (M+1).

Step C: (1R)-1-[6-(Trifluoromethyl)-3-pyridinyl]ethanamine

To a solution of2-methyl-N-{(1R)-1-[6-(trifluoromethyl)-3-pyridinyl]ethyl}-2-propanesulfinamide(41.6 g, 141 mmol) in methyl alcohol (470 mL) at 0° C. was addedhydrogen chloride (4.0 Min dioxane; 106 mL, 424 mmol). After 30 min, themixture was concentrated to dryness. The residue was recrystallizedusing ethyl alcohol (15 mL) and ether (40 mL). The white solid wasfiltered and dried under reduced pressure to give the hydrochloride saltof the title compound (26.3 g). MS 191.2 (M+1). ¹H NMR (500 MHz, CD₃OD): δ 8.83 (d, J=2.2 Hz, 1H); 8.17 (d, J=8.2 Hz, 1H); 7.93 (d, J=8.2Hz, 1H); 4.69 (q, J=6.9 Hz, 1H); 1.70 (d, J=6.9 Hz, 3H).

(1R)-1-[1-Oxido-6-(trifluoromethyl)-3-pyridinyl]ethanamine Step A:tert-Butyl{(1R)-1-[6-(trifluoromethyl)-3-pyridinyl]ethyl}carbamate

To a solution of (1R)-1-[6-(trifluoromethyl)pyridin-3-yl]ethanaminehydrochloride salt (0.554 g, 0.21 mmol) in dichloromethane (7.0 mL) wereadded di-tert-butyl dicarbonate (0.506 g, 2.32 mmol) and triethylamine(0.969 mL, 6.95 mmol). The reaction mixture was stirred at ambienttemperature for 4 h. Saturated aqueous ammonium chloride was added. Themixture was extracted with dichloromethane (3×). The combined organicsextracts were washed with brine, dried over magnesium sulfate, filteredand concentrated to give the title compound which was used directly inStep B (0.626 g).

Step B:tert-Butyl{(1R)-1-[1-oxido-6-(trifluoromethyl)-3-pyridinyl]ethyl}carbamate

To a solution oftert-butyl{(1R)-1-[6-(trifluoromethyl)-3-pyridinyl]ethyl}carbamate(0.626 g, 2.157 mmol) in chloroform (10.0 mL) were added2,6-di-tert-butyl-4-methylphenol (24 mg, 0.108 mmol) and3-chloroperbenzoic acid (0.665 g, 2.70 mmol). The reaction mixture wasstirred at 50° C. for 48 h. The reaction mixture was cooled to ambienttemperature. Saturated aqueous sodium thiosulfate and saturated aqueoussodium bicarbonate were added. The mixture was extracted withdichloromethane (3×). The combined organics extracts were washed withbrine, dried over magnesium sulfate, filtered and concentrated.Purification by silica gel chromatography (75% hexanes/ethylacetate→100% ethyl acetate) gave the title compound (140 mg). MS 307.0(M+1).

Step C: (1R)-1-[1-Oxido-6-(trifluoromethyl)-3-pyridinyl]ethanaminehydrochloride

To a solution oftert-butyl{(1R)-1-[1-oxido-6-(trifluoromethyl)-3-pyridinyl]ethyl}carbamate(140 mg, 0.457 mmol) in dioxane (2 mL) was added hydrogen chloride (4.0M in dioxane; 0.343 mL, 1.371 mmol). The reaction mixture was stirredfor 4 h. The reaction mixture was concentrated to dryness to give thehydrochloride salt of the title compound (118 mg). MS 207.1 (M+1).

(1R)-1-(3-Methyl-1,2,4-oxadiazol-5-yl)ethanamine Step A:tert-Butyl[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-1 ethyl]carbamate

To a solution of N-(tert-butoxycarbonyl)-D-alanine (20 g, 106 mmol),acetamide oxime (17.3 g, 234 mmol) in 120 mL of 1,4-dioxane and 30 mL ofN,N-dimethylformamide were added EDC (44.8 g, 234 mmol). The mixture washeated at 60° C. for 4 h then at 100 for 16 h. After cooling to ambienttemperature, 300 mL of ethyl acetate was added. The mixture was washedwith aqueous saturated sodium bicarbonate (2×). The combined organicextracts were dried over magnesium sulfate, filtered and concentrated.The residue was purified by silica gel chromatography (100%dichloromethane→90% dichloromethane/methanol) to give puretert-butyl[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]carbamate (6.0g). MS 172.1 ((M-t-butyl carbamate+H)+1).

Step B: (1R)-1-(3-Methyl-1,2,4-oxadiazol-5-yl)ethanamine

To a solution oftert-butyl[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]carbamate (6.0 g,26.4 mmol) in dioxane (40 mL) was added 4 M hydrochloric acid in dioxane(30 mL). The reaction mixture was stirred for 16 h. The solution wasconcentrated and dried by vacuum to give hydrochloride salt of(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethanamine (5.1 g). ¹H NMR (500MHz, CD₃OD): δ 490-4.83 (m, 1H); 2.41 (s, 3H); 1.72 (d, J=7.0 Hz, 3H).MS 128.2 (M+1).

(1R)-1-(5-Fluoropyridin-2-yl)ethanamine Step A: Ethyl5-fluoropyridine-2-carboxylate

To a degassed solution of ethyl alcohol (400 mL) in a Parr steel bombwas added sodium acetate (43.3 g, 528 mmol), 2-bromo-5-fluoropyridine(20 g, 114 mmol), 1,1′-bis(diphenylphosphino)ferrocene (2.27 g, 4.09mmol) and palladium(II) acetate (204 mg, 0.91 mmol). The vessel was putunder nitrogen and sealed with Parr top. The atmosphere was displacedwith carbon monoxide gas and the pressure was adjusted to 300 psi. Themixture was heated to 90° C. After 3 h, the pressure dropped to below100 psi. The vessel was cooled to ambient temperature and the reactionwas repressurized with carbon monoxide to 300 psi. The vessel was heatedto 90° C. for an additional 4 h. The vessel was cooled to ambienttemperature and the remaining carbon monoxide was vented. The mixturewas concentrated to half of the volume. Ethyl acetate (500 mL) and water(300 mL) were added. The organic layer was isolated and the aqueouslayer was extracted with ethyl acetate (2×). The combined organicextracts were washed with brine, dried over sodium sulfate, filtered andconcentrated. Purification by silica gel chromatography (100%hexanes→70% hexanes/ethyl acetate) gave the title compound. MS 170.0(M+1).

Step B: 5-Fluoropyridine-2-carbaldehyde

To a solution of ethyl 5-fluoropyridine-2-carboxylate (25 g, 148 mmol)in tetrahydrofuran (250 mL) at −78° C. was added dropwisediisobutylaluminum hydride (1.0 M in hexanes; 296 mL, 296 mmol). After 1h, the reaction was quenched with ethyl alcohol (10 mL). Saturatedaqueous sodium potassium tartrate tetrahydrate (1.3 L) was added and theaqueous layer was extracted with ethyl acetate (2×). The combinedorganic extracts were washed with brine, dried over sodium sulfate,filtered. The solution mixture (1.4 L) was carried onto the next stepwithout concentration. MS 125.9 (M+1).

Step C:N-[(1E)-(5-Fluoropyridin-2-yl)methylene]-2-methylpropane-2-sulfinamide

To a solution of 5-fluoropyridine-2-carbaldehyde (18.49 g, 148 mmol) inethyl acetate (850 mL), tetrahydrofuran (250 mL) and hexanes (300 mL)were added (R)-(+)-2-methyl-2-propanesulfinamide (19.71 g, 163 mmol) andanhydrous copper(II) sulfate (59.0 g, 370 mmol). The mixture was stirredat ambient temperature. After 18 h, the mixture was filtered throughCelite. The filtered cake was washed with ethyl acetate and the filtratewas concentrated. Purification by silica gel chromatography (100%dichloromethane→98% dichloromethane/methanol) gave the title compound.

Step D:N-[(1R)-1-(5-Fluoropyridin-2-yl)ethyl]-2-methylpropane-2-sulfinamide

To a solution ofN-[(1E)-(5-fluoropyridin-2-yl)methylene]-2-methylpropane-2-sulfinamide(52.12 g, 228 mmol) in dichloromethane (1000 mL) at −78° C. was addedmethylmagnesium bromide (3.0 M in tetrahydrofuran; 198 mL, 594 mmol).The mixture was allowed to warm to ambient temperature. After 30 min,the mixture was cooled down to −78° C. and was quenched with saturatedaqueous ammonium chloride (100 mL). The mixture was allowed to warm toambient temperature. The organic layer was separated and the aqueouslayer was extracted with dichloromethane (3×). The combined organicextracts were washed with brine, dried over sodium sulfate, filtered andconcentrated. Purification by silica gel chromatography (100% ethylacetate) gave the title compound. MS 245 (M+1).

Step E: (1R)-1-(5-Fluoropyridin-2-yl)ethanamine

To a solution ofN-[(1R)-1-(5-fluoropyridin-2-yl)ethyl]-2-methylpropane-2-sulfinamide(34.3 g, 140 mmol) in methyl alcohol (700 mL) at 0° C. was addedhydrogen chloride (4.0 M in dioxane; 105 mL, 421 mmol). After 30 min,the mixture was concentrated to dryness. The residue was recrytalizedusing ethyl alcohol (15 mL) and ether (40 mL). The white solid wasfiltered and dried under reduced pressure to give the hydrochloride saltof the title compound. MS 141.1 (M+1).

(1R)-1-(5-Fluoro-1-oxidopyrindin-2-yl)ethanamine Step A:tert-Butyl[(1R)-1-(5-fluoropyridin-2-yl)ethyl]carbamate

To a solution of the toluene sulfonic acid salt of(1R)-1-(5-fluoropyridin-2-yl)ethanamine (7.5 g, 24.0 mmol) indichloromethane (96 mL) at 0° C. was added triethylamine (7.03 mL, 50.0mmol) and di-tert-butyl dicarbonate (6.13 mL, 26.4 mmol). The mixturewas allowed to warm to ambient temperature. After 16 hours, saturatedaqueous sodium bicarbonate was added. The organic layer was isolated andthe aqueous layer was extracted with dichloromethane (2×). The combinedorganic extracts were washed with brine, dried over magnesium sulfate,and filtered. Concentration gave the title compound (7.72 g). MS 241.1(M+1).

Step B: tert-Butyl[(1R)-1-(5-fluoro-1-oxidopyridin-2-yl)ethyl]carbamate

To a solution of tert-butyl[(1R)-1-(5-fluoropyridin-2-yl)ethyl]carbamate(5.77 g, 24.0 mmol) in dichloromethane (96 mL) was added3-chloroperbenzoic acid (6.51 g, 26.4 mmol). After 4.5 h, excess3-chloroperbenzoic acid (0.59 g, 2.6 mmol) was added. After 72 h,saturated aqueous sodium sulfite was added. After 1 h, saturated aqueoussodium bicarbonate was added. The organic layer was isolated and theaqueous layer was extracted with dichloromethane (2×). The combinedorganic extracts were washed with brine, dried over magnesium sulfate,filtered, and concentrated. Purification by silica gel chromatography(100% dichloromethane 90% dichloromethane/methanol with 1% ammoniumhydroxide) gave the title compound (5.45 g). MS 257.1 (M+1).

Step C: (1R)-1-(5-Fluoro-1-oxidopyrindin-2-yl)ethanamine

To a solution oftert-butyl[(1R)-1-(5-fluoro-1-oxidopyridin-2-yl)ethyl]carbamate (1.47 g,5.74 mmol) in dichloromethane (28.7 mL) was added 4 M hydrochloric acidin 1,4-dioxane (43.0 mL, 172 mmol). After 2 h, concentration gave thetitle compound as a hydrochloride salt (1.396 g). MS 157.1 (M+1). ¹H NMR(500 MHz, CD₃OD): δ 8.55 (dd, J=4.3, 2.4 Hz, 1H); 7.70 (dd, J=9.0, 6.7Hz, 1H); 7.52 (ddd, J=9.1, 7.1, 2.4 Hz, 1H); 4.80 (q, J=7.0 Hz, 1H);1.74 (d, J=7.0 Hz, 3H).

(1R)-1-(5-Methyl-1,2,4-oxadiazol-3-yl)ethanamine Step A:Benzyl[(1R)-1-cyanoethyl]carbamate

To a solution of benzyl[(1R)-2-amino-1-methyl-2-oxoethyl]carbamate (10g, 45 mmol) in 50 mL of N,N-dimethylformamide was added2,4,6-trichloro-1,3,5-triazine (4.15 g, 22.5 mmol). After 2 h, 100 mL ofwater was added and the mixture was filtered. The solids were washedwith 100 mL aqueous sodium bicarbonate (2×) and dried under vacuum togive pure benzyl[(1R)-1-cyanoethyl]carbamate (7.2 g). MS 205.2 ((M+1).

Step. B: Benzyl[(1R,2Z)-2-amino-2-(hydroximino 1-methylethyl]carbamate

To a solution of benzyl[(1R)-1-cyanoethyl]carbamate (2.52 g, 12.3 mmol)in ethanol (30 ml) was added hydroxylamine hydrochloride salt (0.90 g,13.0 mmol) and triethylamine (3.43 ml, 24.6 mmol) and the mixture heatedto 75° C. After 16 h, the solution was concentrated and the residue wasdissolved in 200 mL of dichloromethane. The mixture was washed with 100ink, of saturated aquous sodium bicarbonate (2×) and saturated aqueoussodium chloride (100 mL). The combined organic extracts were dried oversodium sulfate, filtered and concentrated to givebenzyl[(1R,2Z)-2-amino-2-(hydroxyimino)-1-methylethyl]carbamate (2.9 g).MS 238.2 (M+1).

Step. C: Benzyl[(1R)-1-(5-methyl-1,2,4-oxadiazol-3-yl)ethyl]carbamate

To a solution ofbenzyl[(1R,2Z)-2-amino-2-(hydroxyimino)-1-methylethyl]carbamate (2.25 g,9.48 mmol) in dioxane (80 ml) was added 1-acetyl-1H-imidazole (3.13 g,28.5 mmol) and the mixture heated to 90° C. After 16 h, the solution wasconcentrated and the residue was dissolved in 200 mL of dichloromethane.The mixture was washed with 100 mL of aquous saturated sodiumbicarbonate (2×) and saturated aqueous sodium chloride (100 mL). Theorganic layer was dried over sodium sulfate, filtered and concentrated.The residue was purified by silica gel chromatography (100%dichloromethane→95% dichloromethane/methanol) to give the title compound(1.1 g). MS 262.1 (M+1).

Step D: (1R)-1-(5-Methyl-1,2,4-oxadiazol-3-yl)ethanamine

To a solution ofbenzyl[(1R)-1-(5-methyl-1,2,4-oxadiazol-3-yl)ethyl]carbamate (1.10 g,4.21 mmol) in dichloromethane (40 mL) was added 1 M boron trichloridesolution in dichloromethane (21.1 mL, 21.1 mmol) at 0° C. The reactionmixture was allowed to warm from 0° C. to 20° C. over 4 h. The solutionwas quenched by 5 ml of methanol at 0° C. After warming to ambienttemperature, the mixture was concentrated and the residue was washedwith 100 mL of diethyl ether (2×) to give the hydrochloride salt of(1R)-1-(5-methyl-1,2,4-oxadiazol-3-yl)ethanamine was obtained as solid(0.84 g). ¹H NMR (500 MHz, CD₃OD): δ 4.70-4.61 (m, 1H); 2.63 (s, 3H);1.67 (d, J=6.9 Hz, 3H).

(1R)-1-[2-(Trifluoromethyl)pyrimidin-5-yl]ethanamine Step A: Ethyl2-(trifluoromethyl)pyrimidine-5-carboxylate

To a solution of ethyl4-chloro-2-(trifluoromethyl)pyrimidine-5-carboxylate (30.2 g, 119.0mmol) in ethanol (594 mL) under nitrogen were added palladium (10% oncarbon, 50% water wet; 2.58 g, 1.21 mmol) and diisopropylethylamine(50.0 mL, 286.0 mmol). The mixture stirred under hydrogen (1 atm). After6 h, the mixture was filtered with Celite. The filtrate was concentratedand ethyl acetate was added. The mixture was washed with saturatedaqueous sodium bicarbonate (2×), saturated aqueous sodium chloride,dried over sodium sulfate, filtered and concentrated to give the titlecompound (25.6 g). MS 221.1 (M+1).

Step B: 2-(Trifluoromethyl)pyrimidine-5-carbaldehyde

To a solution of ethyl 2-(trifluoromethyl)pyrimidine-5-carboxylate (25.5g, 116.0 mmol) in dichloromethane (580 mL) at −78° C. was slowly addeddiisobutylaluminium hydride (1.0 M; 130.0 mL, 130.0 mmol). The mixturewas stirred at −78° C. After 2 h, the mixture was quenched via slowaddition of hydrochloric acid (2.0 M). The mixture was allowed to warmto ambient temperature. The mixture was extracted with diethyl ether(3×). The combined organic extracts was dried over sodium sulfate,filtered and concentrated to give the title compound (28.2 g).

Step C:2-Methyl-N-{(1Z)-[2-(trifluoromethyl)pyrimidin-5-yl]methylene}propane-2-sulfinamide

To a solution of 2-(trifluoromethyl)pyrimidine-5-carbaldehyde (27.2 g,99 mmol) in dichloroethane (250 mL) was added(R)-(+)-2-methyl-2-propanesulfinamide (13.3 g, 109.0 mmol) andcopper(II) sulfate (31.5 g, 197.0 mmol). The mixture was heated to 50°C. After 18 h, the mixture was cooled to ambient temperature andfiltered through a pad of silica gel. The filtered cake was washed withdichloromethane and the filtrate was concentrated to give the titlecompound (27.3 g). MS 224 [(M+1)−56].

Step D:2-Methyl-N-{(1R)-1-[2-(trifluoromethyl)pyrimidin-5-yl]ethyl}propane-2-sulfinamide

To a solution of2-methyl-N-{(1Z)-[2-(trifluoromethyl)pyrimidin-5-yl]methylene}propane-2-sulfinamide(14.3 g, 51.2 mmol) in toluene (260 mL) at −70° C. was addedmethyllithium (1.6 M; 35.0 mL, 56.0 mmol). The mixture was stirred at−70° C. for 15 min. The mixture was quenched with saturated aqueousammonium chloride and the reaction was allowed to warm to ambienttemperature. The mixture was extracted with dichloromethane (3×). Thecombined organic extracts was dried over Na₂SO₄, filtered andconcentrated. Purification by silica gel chromatography (100%hexanes→35% hexanes/ethyl acetate then 100% ethyl acetate—94% ethylacetate/methanol) gave the title compound (7.23 g). MS 240.0 [(M+1)−56].

Step E: (1R)-1-[2-(Trifluoromethyl)pyrimidin-5-yl]ethanamine

To a solution of2-methyl-N-{(1R)-1-[2-(trifluoromethyl)pyrimidin-5-yl]ethyl}propane-2-sulfinamide(7.23 g, 24.5 mmol) in methanol (100 mL) was added hydrogen chloride(4.0 Min dioxane; 18.5 mL, 74.0 mmol). The mixture was stirred atambient temperature. After 1 h, the mixture was concentrated to give thetitle compound (4.6 g).

Example 1.2

1-Methyl-3-phenyl-N-{(1R)-1-[6-(trifluoromethyl)pyridin-3-yl]ethyl}-1H-thieno[2,3-c]pyrazole-5-carboxamide

To a solution of 1-methyl-3-phenyl-1H-thieno[2,3-c]pyrazole-5-carboxylicacid (25.0 mg, 0.10 mmol),(1R)-1-[6-(trifluoromethyl)pyridin-3-yl]ethanamine (22.1 mg, 0.12 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (32.5 mg,0.17 mmol), 1-hydroxy-7-azabenzotriazole (6.6 mg, 48.0 μmol) inN,N-dimethylformamide (1 mL) was added triethylamine (54.0 μL, 0.39mmol). The mixture was allowed to stir at 50° C. After 18 h, the mixturewas cooled to ambient temperature and water (100 μL) and trifluoroaceticacid (100 μL) were added. Purification by reverse phase HPLC (C-18, 95%water/acetonitrile→5% water/acetonitrile with 0.1% trifluoroacetic acid)gave the trifluoroacetate salt of the title compound (38 mg): HRMS [M+1]found=431.1166. ¹H NMR (500 MHz, DMSO-d₆): δ 9.07 (1H, d, J=7.27 Hz),8.83 (1H, s), 8.37 (1H, s), 8.09 (1H, d, J=8.25 Hz), 7.95-7.89 (3H, m),7.52 (2H, t, J=7.57 Hz), 7.41 (1H, t, J=7.37 Hz), 5.29-5.22 (1H, m),4.01 (3H, s), 1.58 (3H, d, J=7.06 Hz).

Example 1.49

3-(2,4-Difluorophenyl)-1-(2-hydroxyethyl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1H-thieno[2,3-c]pyrazole-5-carboxamideStep A: 3,5-Dibromo-1-(4-methoxybenzyl)-1H-pyrazole-4-carbaldehyde

3,5-Dibromo-1H-pyrazole-4-carbaldehyde (987.9 mg, 3.89 mmol) andpotassium carbonate (1.613 g, 11.67 mmol) were dissolved inN,N-dimethylformamide (39 mL) at 25° C. under Ar. 4-methoxybenzylbromide (617 μl, 4.28 mmol) was added dropwise. The reaction mixture wasallowed to stir for 2 h 10 min. The reaction was stopped, quenched byaddition of saturated aqueous ammonium chloride (30 mL), and the mixtureextracted with ethyl acetate (3×30 mL). The combined organic phases werewashed with saturated aqueous sodium chloride (1×20 mL), dried (sodiumsulfate), filtered, and the solvent evaporated under reduced pressure.The crude product was purified by flash chromatography (RediSep SiO₂,120 g column) on a CombiFlash Rf purification system eluting with ethylacetate-hexanes (0-55%). The title compound (L3342 g, 3.57 mmol, 92%yield) was recovered as a colorless, very viscous oil. LC-MS[M+1]=373.1.

Step B: Ethyl3-bromo-1-(4-methoxybenzyl)-1H-thieno[2,3-c]pyrazole-5-carboxylate

3,5-Dibromo-1-(4-methoxybenzyl)-1H-pyrazole-4-carbaldehyde (1.3301 g,3.56 mmol), ethyl thioglycolate (0.419 ml, 3.73 mmol), and sodiumcarbonate (0.761 g, 7.18 mmol) were dissolved in ethanol (35 ml) at 25°C. under Ar in a sealed heavy-walled reaction vessel. The reactionmixture was warmed to 80° C. and allowed to stir for 4 h. The reactionwas stopped, cooled to room temperature, quenched by addition ofsaturated aqueous ammonium chloride (40 mL), and the mixture extractedwith ethyl acetate (3×30 mL). The combined organic phases were washedwith saturated aqueous sodium chloride (1×20 mL), dried (sodiumsulfate), filtered, and the solvent evaporated under reduced pressure.The crude product was purified by flash chromatography (RediSep SiO₂,120 g column) on a CombiFlash Rf purification system eluting with ethylacetate-hexanes (0-25%). The title compound (1.2063 g, 3.05 mmol, 86%yield) was recovered as a white solid. LC-MS [M+1]=395.2.

Step C: Ethyl3-(2,4-difluorophenyl)-1-(4-methoxybenzyl)-1H-thieno[2,3-c]pyrazole-5-carboxylate

Ethyl 3-bromo-1-(4-methoxybenzyl)-1H-thieno[2,3-c]pyrazole-5-carboxylate(1.1422 g, 2.89 mmol), 2,4-difluorophenylboronic acid (1.867 g, 11.82mmol), sodium carbonate (1.614 g, 15.23 mmol), and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.426 g,0.582 mmol) were dissolved in dioxane (14.45 ml) at 25° C. under Ar in asealed heavy-walled reaction vessel. The reaction mixture was heated to80° C. and allowed to stir for 2 h. Cesium carbonate (1.52 g, 4.67 mmol)was added. The reaction was allowed to stir for 2 h. The reaction wasstopped, cooled to room temperature, quenched by addition of saturatedaqueous ammonium chloride (20 mL), and the mixture extracted with ethylacetate (3×20 mL). The combined organic phases were washed withsaturated aqueous sodium chloride (1×15 mL), dried (sodium sulfate),filtered, and the solvent evaporated under reduced pressure. The crudeproduct was purified by flash chromatography (RediSep SiO₂, 120 gcolumn) on a CombiFlash purification system eluting with ethylacetate-hexanes (0-15%). The title compound (1.0147 g, 2.368 mmol, 82%yield) was recovered as a white solid. LC-MS [M+1]=429.3

Step D: Ethyl3-(2,4-difluorophenyl)-1H-thieno[2,3-c]pyrazole-5-carboxylate

Ethyl3-(2,4-difluorophenyl)-1-(4-methoxybenzyl)-1H-thieno[2,3-c]pyrazole-5-carboxylate(1.0103 g, 2.358 mmol) was dissolved in a mixture of 1,2-dichloroethane(11.79 ml) and trifluoroacetic acid (11.79 ml) at 25° C. in a sealedheavy-walled reaction vessel. The reaction mixture was heated to 100° C.and allowed to stir for 2 h. The reaction was stopped, cooled to roomtemperature, and concentrated under reduced pressure. The residue wastaken up in ethyl acetate (20 mL), washed with saturated aqueous sodiumbicarbonate (1×15 mL) and saturated aqueous sodium chloride (1×15 mL),dried (sodium sulfate), and concentrated under reduced pressure. Thecrude product was purified by flash chromatography (RediSep SiO₂, 120 gcolumn) on a CombiFlash purification system eluting with ethylacetate-hexanes (0-60%). The title compound (719.0 mg, 2.332 mmol, 99%yield) was recovered as a white solid. LC-MS [M+1]=309.3.

Step E: 3-(2,4-Difluorophenyl)-1H-thieno[2,3-c]pyrazole-5-carboxylicacid

Ethyl 3-(2,4-difluorophenyl)-1H-thieno[2,3-c]pyrazole-5-carboxylate(714.9 mg, 2.319 mmol) was dissolved in tetrahydrofuran (35 mL)/methanol(12 mL) at 25° C. Sodium hydroxide (9.28 mL, 9.28 mmol) was added. Thereaction mixture was heated to 50° C. and allowed to stir for 18 h. Thereaction was stopped, cooled to room temperature, quenched withconcentrated hydrochloric acid (0.767 mL), and concentrated underreduced pressure. The title compound, with 4 equivalents of sodiumchloride, (1.1613 g, 2.259 mmol, 97% yield) was recovered as a lightyellow/white solid. LC-MS [M+1]=281.2.

Step F:3-(2,4-Difluorophenyl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1H-thieno[2,3-c]pyrazole-5-carboxamide

3-(2,4-Difluorophenyl)-1H-thieno[2,3-c]pyrazole-5-carboxylic acid (964.9mg, 1.877 mmol, with 4 equivalents of sodium chloride),L-(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethanamine bis-hydrochloride(462.3 mg, 2.311 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (725.4 mg, 3.78 mmol), 1-hydroxy-7-azabenzotriazole (129.6mg, 0.952 mmol), and N-methylmorpholine (0.826 mL, 7.51 mmol) weredissolved in N,N-dimethylformamide (18 mL) at 25° C. The reactionmixture was heated to 50° C. and allowed to stir for 10 min. Thereaction was stopped, cooled to room temperature, quenched by additionof saturated aqueous ammonium chloride (15 mL), and extracted with ethylacetate (3×15 mL). The combined organic phases were washed withsaturated aqueous ammonium chloride (2×15 mL) and saturated aqueoussodium chloride (1×15 mL), dried (sodium sulfate), filtered, and thesolvent evaporated under reduced pressure. The crude product waspurified by flash chromatography (RediSep SiO₂, 120 g column) on aCombiFlash purification system eluting with ethyl acetate-hexanes(0-100%). The title compound (556.0 mg, 1.428 mmol, 76% yield) wasrecovered as a light yellow/white solid. LC-MS [M-1-1]=390.2

Step G:3-(2,4-Difluorophenyl)-1-(2-hydroxyethyl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1H-thieno[2,3-c]pyrazole-5-carboxamide

3-(2,4-Difluorophenyl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1H-thieno[2,3-c]pyrazole-5-carboxamide(203.5 mg, 0.523 mmol) and potassium carbonate (331.5 mg, 2.40 mmol)were dissolved in N,N-dimethylformamide (5.20 ml) at 25° C.2-Iodoethanol (69 μL, 0.885 mmol) was added. The reaction was heated to50° C. and stirred for 4 h. The reaction was stopped, cooled to roomtemperature, quenched by addition of saturated aqueous ammonium chloride(10 mL), and extracted with ethyl acetate (3×15 mL). The combinedorganic phases were washed with saturated aqueous ammonium chloride(2×10 mL) and saturated aqueous sodium chloride (1×10 mL), dried (sodiumsulfate), filtered, and the solvent evaporated under reduced pressure.The crude product was purified by flash chromatography (RediSep SiO₂, 40g column) on a CombiFlash purification system eluting with ethylacetate-hexanes (0-100%). The title compound (129.6 mg, 0.299 mmol,57.2% yield) was recovered as a light yellow/white solid. The undesiredregioisomer,3-(2,4-difluorophenyl)-2-(2-hydroxyethyl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-2H-indazole-6-carboxamide(33.1 mg, 0.076 mmol, 14.61% yield), was recovered as a lightyellow/white solid. Title compound: HRMS [M+1] found=434.1091. ¹H NMR(400 MHz, CDCl₃): δ 7.99-7.89 (m, 1H); 7.61 (d, J=3.7 Hz, 1H); 7.01-6.91(m, 2H); 6.80 (d, J=7.8 Hz, 1H); 5.60-5.51 (m, 1H); 4.39-4.34 (m, 2H);4.15 (d, J=5.4 Hz, 2H); 2.90 (s, 1H); 2.40 (s, 3H); 1.70 (d, J=7.1 Hz,3H). Regioisomer: HRMS [M+1] found 434.1095.

Example 1.84&85

3-2,4-Difluorophenyl)-1-(3-hydroxybutan-2-yl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1H-thieno[2,3-c]pyrazole-5-carboxamideStep A: Ethyl3-(2,4-difluorophenyl)-1-(3-hydroxybutan-2-yl)-1H-thieno[2,3-c]pyrazole-5-carboxylate

To a solution of ethyl3-(2,4-difluorophenyl)-1H-thieno[2,3-c]pyrazole-5-carboxylate (200 mg,0,649 mmol) and potassium carbonate (448 mg, 3.24 mmol) inN,N-dimethylformamide (6.5 mL) was added trans-dimethyloxirane (140 mg,1.95 mmol). The reaction mixture was stirred at 100° C. for 18 hr. Themixture was cooled to ambient temperature. Purification by reverse phaseHPLC (C-18, 95% water/acetonitrile→5% water/acetonitrile with 0.05%ammonium hydroxide) gave the title compound (134 mg). LC-MS [M+1]=381.2.

Step B:3-(2,4-Difluorophenyl)-1-(3-hydroxybutan-2-yl)-1H-thieno[2,3-c]pyrazole-5-carboxylicacid

To a solution of ethyl3-(2,4-difluorophenyl)-1-(3-hydroxybutan-2-yl)-1H-thieno[2,3-c]pyrazole-5-carboxylate(134 mg, 0.352 mmol) in methanol (3.5 mL) was added sodium hydroxide (1M, 1.06 mL, 1.06 mmol). The mixture was stirred at 50° C. for 18 hr.Hydrochloric acid (6M, 0.176 mL, 1.06 mmol) was added. The mixture wasconcentrated to give the title compound with three equivalents of sodiumchloride (181 mg): LC-MS [M+1]=353.3.

Step G:3-(2,4-Difluorophenyl)-1-(3-hydroxybutan-2-yl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1H-thieno[2,3-c]pyrazole-5-carboxamide

To a solution of3-(2,4-difluorophenyl)-1-(3-hydroxybutan-2-yl)-1H-thieno[2,3-e]pyrazole-5-carboxylicacid with three equivalents of sodium chloride (140 mg, 0.265 mmol),(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethanamine hydrochloride (63.7 mg,0.318 mmol, and N-methylmorpholine (33.7 μL, 0.307 mmol) inN,N-dimethylformamide (1.3 mL) was addedN-[2-(dimethylamino)ethyl]-N-ethylcarbodiimide hydrochloride (89 mg,0.464 mmol) and 1-hydroxy-7-azabenzotriazole (18.1 mg, 0.133 mmol). Thereaction mixture was stirred at 50° C. for 1 h. Purification by reversephase HPLC (C-18, 95% water/acetonitrile→5% water/acetonitrile with0.05% ammonium hydroxide) gave the title compound (105 mg). LC-MS [M+1]found=462.4. The diastereomers were separated using a ChiralPak ADcolumn (40% hexane/60% ethanol, diethylamine modifier, 5 cm×50 cm).First eluting diastereomer (38.1 mg): ¹H NMR. (400 MHz, DMSO-d₆) δ: 9.34(1H, d, J=7.37 Hz), 8.06 (1H, d, J=3.55 Hz), 7.96 (1H, td, J=8.74, 6.61Hz), 7.49 (1H, ddd, J=11.26, 9.19, 2.61 Hz), 7.24 (1H, td, J=8.47, 2.61Hz), 5.39-5.28 (1H, m), 5.17 (1H, d, J=5.30 Hz), 4.44-4.37 (1H, m),4.07-3.96 (1H, m), 2.34 (3H, s), 1.57 (6H, dd, J=15.26, 7.01 Hz), 1.01(3H, d, J=6.29 Hz). Second eluting diastereomer (38.4 mg): ¹H NMR δ (400MHz, DMSO-d₆): 9.34 (1H, d, J=7.35 Hz), 8.07 (1H, d, J=3.55 Hz), 7.96(1H, td, J=8.72, 6.64 Hz), 7.49 (1H, ddd, J=11.25, 9.21, 2.61 Hz), 7.24(1H, td, J=8.50, 2.57 Hz), 5.40-5.28 (1H, m), 5.17 (1H, d, J=4.87 Hz),4.45-4.36 (1H, m), 4.07-3.96 (1H, m), 2.33 (3H, s), 1.58 (6H, dd,J=15.83, 7.01 Hz), 1.01 (3H, d, J=6.29 Hz).

Example 1.98

3-(2,4-Difluorophenyl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1-(pyrazin-2-yl)-1H-thieno[2,3-c]pyrazole-5-carboxamideStep A Ethyl3-(2,4-difluorophenyl)-1-(pyrazin-2-yl)-1H-thieno[2,3-c]pyrazole-5-carboxylate

Ethyl 3-(2,4-difluorophenyl)-1H-thieno[2,3-c]pyrazole-5-carboxylate(82.0 mg, 0.266 mmol), 2-fluoropyrazine (134.6 mg, 1.372 mmol) andcesium carbonate (263.7 mg, 0.809 mmol) were dissolved inN,N-dimethylformamide (2.60 mL) at 25° C. under Ar. The reaction mixturewas heated to 80° C. allowed to stir for 2 h. The reaction was stopped,cooled to room temperature, quenched by addition of saturated aqueousammonium chloride (10 mL), and the mixture extracted with ethyl acetate(3×15 mL). The combined organic phases were washed with saturatedaqueous ammonium chloride (2×10 mL) and saturated aqueous sodiumchloride (1×10 mL), dried (sodium sulfate), filtered, and the solventevaporated under reduced pressure. The crude product was purified byflash chromatography (RediSep SiO₂, 40 g column) on a CombiFlash Rfpurification system eluting with ethyl acetate-hexanes (0-50%). Thetitle compound (55.3 mg, 0.143 mmol, 53.8% yield) was recovered as alight yellow/white solid. LC-MS [M+1] 387.3.

Step B:3-(2,4-Difluorophenyl)-1-(pyrazin-2-yl)-1H-thieno[2,3-c]pyrazole-5-carboxylicacid

Ethyl3-(2,4-difluorophenyl)-1-(pyrazin-2-yl)-1H-thieno[2,3-c]pyrazole-5-carboxylate(54.0 mg, 0.140 mmol) was dissolved in tetrahydrofuran (900 μl) andmethanol (600 μl) at 25° C. 1M sodium hydroxide (559 μl, 0.559 mmol) wasadded and the reaction mixture heated to 50° C., and stirred for 12 h.The reaction was stopped, cooled to room temperature, quenched byaddition of concentrated hydrochloric acid (46.2 μL), and concentratedunder reduced pressure. The title compound with 4 equivalents of sodiumchloride (87.2 mg, 0.139 mmol, 99% yield) was recovered as a lightyellow/white solid. LC-MS [M+1]=359.2.

Step C:3-(2,4-Difluorophenyl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1-(pyrazin-2-yl)-1H-thieno[2,3-c]pyrazole-5-carboxamide

3-(2,4-Difluorophenyl)-1-(pyrazin-2-yl)-1H-thieno[2,3-c]pyrazole-5-carboxylicacid (43.6 mg, 0.069 mmol, with 4 equivalents of sodium chloride),L-(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethanamine bis-hydrochloride(17.2 mg, 0.086 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (28.5 mg, 0.149 mmol), 1-hydroxy-7-azabenzotriazole (5.2mg, 0.038 mmol), and N-methylmorpholine (31 μl, 0.282 mmol) weredissolved in N,N-dimethylformamide (700 μl) at 25° C. The reactionmixture was heated to 50° C. and allowed to stir for 10 min. Thereaction was stopped, quenched by addition of water (ca 0.2 mL) andtrifluoroacetic acid (ca 0.2 mL). The reaction mixture was purifieddirectly by preparative HPLC (Reverse phase (C-18)), eluting withacetonitrile/water 0.05% ammonium hydroxide, to give the title compound(21.5 mg, 0.046 mmol, 66.3% yield) as a white solid. HRMS [M+1]found=468.1063. ¹H. NMR (500 MHz, CDCl₃): δ 9.45 (s, 1H); 8.54-8.46 (m,2H); 8.20-8.13 (m, 1H); 7.74 (d, J=3.9 Hz, 1H); 7.10-7.04 (m, 1H);7.03-6.97 (m, 1H); 6.89 (d, J=7.8 Hz, 1H); 5.63 (t, J=7.3 Hz, 1H); 2.44(s, 3H); 1.74 (d, J=7.1 Hz, 3H).

Example 3.1

3-(2-Chlorophenyl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1-(4-methylphenyl)-1H-indazole-6-carboxamideStep A: Methyl 1-(4-methylphenyl)-1H-indazole-6-carboxylate

To a solution of methyl 1H-indazole-6-carboxylate (2.0 g, 11.4 mmol) intoluene (11.4 mL) was added 4-bromotoluene (2.33 g, 13.6 mmol), tribasicpotassium phophate (4.8 g, 22.7 mmol), copper iodide (0.12 g, 0.60 mmol)and trans-(1R,2R)—N,N′-bismethyl-1,2-cyclohexanediamine (0.18 mL, 1.14mmol). The reaction mixture was stirred in a sealed tube at 120° C.After 10 min, 4-iodotoluene (3.01 g) was added and the mixture wasstirred at 120° C. for another 2 h. The mixture was cooled to ambienttemperature and quenched with saturated sodium bicarbonate (20 mL). Themixture was extracted with ethyl acetate (3×20 mL). The combined organiclayers were washed with saturated aqueous sodium chloride (1×15 mL),dried over sodium sulfate, filtered and concentrated under reducedpressure to give the title compound (1.76 g): LC-MS [M+1]=267.1.

Step B: Methyl 3-bromo-1-(4-methylphenyl)-1H-indazole-6-carboxylate

Methyl 1-(4-methylphenyl)-1H-indazole-6-carboxylate (2.7075 g, 10.17mmol) was dissolved in acetonitrile (102 ml) at 25° C. under Ar. Bromine(1.886 ml, 36.60 mmol) was added. The reaction mixture was allowed tostir for 60 h. The reaction was stopped, quenched by addition ofsaturated aqueous sodium bicarbonate (40 mL), and the mixture extractedwith ethyl acetate (3×35 mL). The combined organic phases were washedwith saturated aqueous sodium chloride (1×25 mL), dried (sodiumsulfate), filtered, and the solvent evaporated under reduced pressure.The crude product was purified by flash chromatography (RediSep SiO₂,330 g column) on a CombiFlash Rf purification system eluting with ethylacetate-hexanes (0-5%). The title compound (2.8006 g, 7.55 mmol, 74.2%yield) was recovered as a white solid. LC-MS [M+1]=346.1.

Step C: Methyl3-(2-chlorophenyl)-1-(4-methylphenyl)-1H-indazole-6-carboxylate

To a mixture of methyl3-bromo-1-(4-methylphenyl)-1H-indazole-6-carboxylate (0.30 g, 0.87mmol), 2-chlorophenylboronic acid (0.21 g, 1.34 mmol), cesium carbonate(0.86 g, 2.64 mmol), copper(I) chloride (91.6 mg, 0.93 mmol),1,1′-bis(diphenylphosphino)ferrocene (51.6 mg, 0.09 mmol) andpalladium(II) acetate (9.8 mg, 0.04 mmol) under argon was addedN,N-dimethylformamide (4.3 mL). The mixture stirred in a sealed tube at90° C. for 30 min. The mixture was cooled to ambient temperature andquenched with saturated sodium bicarbonate (15 mL). The mixture wasextracted with ethyl acetate (3×15 mL). The combined organic layers werewashed with saturated aqueous sodium chloride (1×10 mL), dried oversodium sulfate, filtered and concentrated under reduced pressure.Purification by silica gel chromatography (100% hexanes→95%hexanes/ethyl acetate) gave the title compound (0.25 g): LC-MS[M+1]=377.2.

Step D: 3-(2-Chlorophenyl)-1-(4-methylphenyl)-1H-indazole-6-carboxylicacid

To a solution of methyl3-(2-chlorophenyl)-1-(4-methylphenyl)-1H-indazole-6-carboxylate (0.25 g,0.66 mmol) in methanol (1.5 mL) and tetrahydrofuran (2.9 mL) was addedsodium hydroxide (1.0 M, 3.98 mL, 3.98 mmol). The mixture was heated to50° C. and stirred for 1.5 h. The mixture was cooled to ambienttemperature and 1.0 M hydrochloric acid (ca 4.0 mL) was added untilpH<3. The mixture was extracted with ethyl acetate (3×10 mL). Thecombined organic layers were washed with saturated aqueous sodiumchloride (1×7 mL), dried over sodium sulfate, filtered and concentratedunder reduced pressure to give the title compound (0.24 g): LC-MS[M+1]=363.2.

Step E:3-(2-Chlorophenyl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1-(4-methylphenyl)-1H-indazole-6-carboxamide

To a solution of3-(2-Chlorophenyl)-1-(4-methylphenyl)-1H-indazole-6-carboxylic acid(20.3 mg, 56.0 μmol) in N,N-dimethylformamide (0.5 mL) were addedhydrochloride salt of (1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethanamine(14.5 mg, 72.0 μmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (23.4 mg, 0.12 mmol), 1-hydroxy-7-azabenzotriazole (4.1mg, 0.03 mmol) and N-methylmorpholine (23.0 mg, 0.23 mmol). The mixturewas heated to 50° C. and stirred for 30 min. The reaction was stopped,cooled to room temperature, and quenched by addition of water (ca 0.2mL) and trifluoroacetic acid (ca 0.2 mL). Purification by reverse phaseHPLC (C-18, 95% water/acetonitrile→5% water/acetonitrile with 0.1%trifluoroacetic acid) gave the trifluoroacetate salt of the titlecompound (27.4 mg): HRMS [M+1] found=472.1551; ¹H NMR (500 MHz, CDCl₃):δ 8.26 (s, 1H); 7.80 (d, J=8.4 Hz, 1H); 7.69-7.62 (m, 3H); 7.60-7.55 (m,2H); 7.46-7.39 (m, 2H); 7.36 (d, J=7.9 Hz, 2H); 7.09 (d, J=7.2 Hz, 1H);5.66-5.59 (m, 1H); 2.44 (s, 3H); 2.40 (s, 3H); 1.72 (d, J=6.7 Hz, 3H).

Example 3.6

3-(2-Chlorophenyl)-1-isopropyl-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1H-indazole-6-carboxamideStep A: Methyl 1-isopropyl-1H-indazole-6-carboxylate

A solution of methyl 1H-indazole-6-carboxylate (1.92 g, 10.9 mmol) andcessium carbonate (7.12 g, 21.8 mmol) in N,N-dimethylformamide (21.8 mL)under argon was heated to 60° C. for 10 min. 2-Iodopropane (1.09 mL,10.9 mmol) was added dropwise. The mixture was stirred at 60° C. for 1.5h. The mixture was cooled to ambient temperature and quenched withsaturated aqueous sodium bicarbonate (30 mL). The mixture was extractedwith ethyl acetate (3×30 mL). The combined organic layer was washed withsaturated aqueous ammonium chloride (3×20 mL), saturated aqueous sodiumchloride (1×20 mL), dried over sodium sulfate, filtered and concentratedunder reduced pressure. Purification by silica gel chromatography (100%hexanes→70% hexanes/ethyl acetate) gave the title compound (1.33 g):LC-MS [M+1]=219.2.

Step B: Methyl 3-bronco-1-isopropyl-1H-indazole-6-carboxylate

To a solution of methyl 1-isopropyl-1H-indazole-6-carboxylate (1.3 g,5.94 mmol) in acetonitrile (50 mL) under argon was added bromine (0.60mL, 11.64 mmol) and acetic acid (0.10 mL). The mixture was stirred atambient temperature for 18 h. The reaction was stopped, and quenchedwith saturated aqueous sodium bicarbonate (30 mL). The mixture wasextracted with ethyl acetate (3×30 mL). The combined organic layer waswashed with saturated aqueous sodium chloride (1×15 mL), dried oversodium sulfate, filtered and concentrated under reduced pressure to givethe title compound (1.74 g): LC-MS [M+1]=297.2.

Step C: Methyl 3-(2-chlorophenyl)-1-isopropyl-1H-indazole-6-carboxylate

To a mixture of methyl 3-bromo-1-isopropyl-1H-indazole-6-carboxylate(0.32 g, 1.08 mmol), 2-chlorophenylboronic acid (0.51 g, 3.29 mmol),cessium carbonate (1.15 g, 3.52 mmol), copper(I) chloride (0.11 g, 1.09mmol), 1,1′-bis(diphenylphosphino)ferrocene (61.1 mg, 0.11 mmol) andpalladium(II) acetate (12.6 mg, 0.06 mmol) under argon was addedN,N-dimethylformamide (4.3 mL). The mixture was stirred in a sealed tubeat 90° C. for 18 h. After 18 h, the mixture was cooled to ambienttemperature and quenched with saturated aqueous sodium bicarbonate (15mL). The mixture was extracted with ethyl acetate (3×15 mL). Thecombined organic layer was washed with saturated aqueous ammoniumchloride (4×10 mL), saturated aqueous sodium chloride (1×10 mL), driedover sodium sulfate, filtered and concentrated under reduced pressure.Purification by silica gel chromatography (100% hexanes→85%hexanes/ethyl acetate) gave the title compound (0.11 g): LC-MS[M+1]=329.2.

Step D: 3-(2-Chlorophenyl)-1-isopropyl-1H-indazole-6-carboxylic acid

To a solution of methyl3-(2-chlorophenyl)-1-isopropyl-1H-indazole-6-carboxylate (57.7 mg, 0.18mmol) in methanol (1.1 mL) and tetrahydrofuran (0.7 mL) was added sodiumhydroxide (1.0 M, 1.10 mL, 1.10 mmol). The mixture was heated to 50° C.and stirred for 18 h. The mixture was cooled to ambient temperature and1.0 M hydrochloric acid (ca 1.1 mL) was added until pH<3. The mixturewas extracted with ethyl acetate (3×10 mL). The combined organic layerwas washed with saturated aqueous sodium chloride (1×7 mL), dried oversodium sulfate, filtered and concentrated to give the title compound(55.0 mg): LC-MS [M+1]=315.0.

Step E:3-(2-Chlorophenyl)-1-isopropyl-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1H-indazole-6-carboxamide

To a solution of 3-(2-chlorophenyl)-1-isopropyl-1H-indazole-6-carboxylicacid (13.3 mg, 42.0 μmol) in N,N-dimethylformamide (0.5 mL) were addedhydrochloride salt of (1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethanamine(10.4 mg, 63.0 μmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (16.2 mg, 85.0 μmol), 1-hydroxy-7-azabenzotriazole (2.9mg, 21.0 μmol) and N-methylmorpholine (18.6 μL, 0.17 mmol). The mixturewas heated to 50° C. and stirred for 30 min. Purification by reversephase HPLC (C-18, 95% water/acetonitrile→5% water/acetonitrile with 0.1%trifluoroacetic acid) gave the bistrifluoroacetate salt of the titlecompound (12 mg): HRMS [M+1] found=424.1530; ¹H NMR (500 MHz, CDCl₃): δ8.12 (s, 1H); 7.75 (d, J=8.4 Hz, 1H); 7.63-7.59 (m, 1H); 7.57-7.53 (m,1H); 7.49 (d, J=8.4 Hz, 1H); 7.39 (dd, J=5.9, 3.5 Hz, 2H); 6.84 (d,J=7.7 Hz, 1H); 5.68-5.61 (m, 1H); 5.03-4.95 (m, 1H); 2.42 (s, 3H); 1.74(d, J=7.0 Hz, 3H); 1.67 (d, J=6.6 Hz, 6H).

Example 3.142

3-(2,6-Difluorophenyl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1-(4-methylphenyl)-1Hindazole-6-carboxamide Step A: Methyl3-(2,6-difluorophenyl)-1-(4-methylphenyl)-1H-indazole-6-carboxylate

Methyl 3-bromo-1-(4-methylphenyl)-1H-indazole-6-carboxylate 173.7 mg,0.503 mmol), 2,6-difluorophenylboronic acid, pinacol ester (183.4 mg,0.764 mmol), Cs₂CO₃ (505.1 mg, 1.550 mmol), copper(I) chloride (52.7 mg,0.532 mmol), 1,1′-bisdiphenylphosphino ferrocene (29.3 mg, 0.053 mmol),and palladium(II) acetate (6.5 mg, 0.029 mmol) were placed in a 10-20 mLmicrowave vial under Ar. N,N-dimethylformamide (2.50 mL) was added andthe vessel sealed. The reaction mixture was heated to 90° C. and allowedto stir for 30 min. The reaction was stopped, quenched by addition ofsaturated aqueous ammonium chloride (15 mL), and extracted with ethylacetate (3×15 mL). The combined organic phases were washed withsaturated aqueous ammonium chloride (1×15 mL), saturated aqueous sodiumchloride (1×10 mL), dried (sodium sulfate), and concentrated underreduced pressure. The crude product was purified by flash chromatography(RediSep SiO₂, 24 g column) on a CombiFlash purification system elutingwith ethyl acetate-hexanes (0-10%). The title compound (102.1 mg, 0.270mmol, 53.6% yield) was recovered as a white solid. LC-MS: [M+1]=379.2.

Step B:3-(2,6-Difluorophenyl)-1-(4-methylphenyl)-1H-indazole-6-carboxylic acid

Methyl3-(2,6-difluorophenyl)-1-(4-methylphenyl)-1H-indazole-6-carboxylate(101.2 mg, 0.267 mmol) was dissolved in methanol (1.30 mL) andtetrahydrofuran (1.90 mL). 1M sodium hydroxide (1.60 mL, 0.267 mmol) wasadded. The reaction mixture was heated to 50° C. and allowed to stir for1.5 h. Reaction stopped, cooled to room temperature, quenched byaddition of 1M hydrochloric acid (ca 1.60 mL) till pH<3, and the solventevaporated under reduced pressure. The title compound (199.5 mg, 0.265mmol, 99% yield, with 6 equivalents of sodium chloride) was recovered asa white solid. LC-MS: [M+1]=365.2.

Step C:3-2,6-Difluorophenyl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1-(4-methylphenyl)-1H-indazole-6-carboxamide

3-(2,6-Difluorophenyl)-1-(4-methylphenyl)-1H-indazole-6-carboxylic acid(30.0 mg, 0.040 mmol, with 6 equivalents of sodium chloride),(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethanamine bis-hydrochloride (12.5mg, 0.062 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (16.2 mg, 0.085 mmol), 1-hydroxy-7-azabenzotriazole (3.4mg, 0.025 mmol), and N-methylmorpholine (20 μl, 0.182 mmol) weredissolved in N,N-dimethylformamide (500 μl) at 25° C. The reactionmixture was heated to 50° C. and allowed to stir for 30 min. Thereaction was stopped and quenched by addition of water (ca 0.1 mL) andtrifluoroacetic acid (ca 0.1 mL). The crude reaction mixture waspurified directly by preparative HPLC (Reverse phase (C-18)), elutingwith acetonitrile/water+0.1% trifluoroacetic acid (5-95%), to afford thetitle compound (16.3 mg, 0.023 mmol, 58.2% yield) as a white solid. HRMS[M+1]=474.1739. ¹H NMR (400 MHz, CDCl₃): δ 8.29 (s, 1H); 7.77 (d, J=8.6Hz, 1H); 7.66 (d, J=8.2 Hz, 2H); 7.61 (dd, J=8.5, 1.4 Hz, 1H); 7.48-7.40(m, 1H); 7.38 (d, J=8.1 Hz, 2H); 7.10 (m, 2H); 6.79 (d, J=7.7 Hz, 1H);5.62 (m, 1H); 2.46 (s, 3H); 2.41 (s, 3H); 1.73 (d, J=7.1 Hz, 3H).

Example 3.208

3-(1,2-Dihydroxypropan-2-yl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1-(4-methylphenyl)-1H-indazole-6-carboxamideStep A: 3-Bromo-1-(4-methylphenyl)-1H-indazole-6-carboxylic acid

Methyl3-(2,6-difluorophenyl)-1-(4-methylphenyl)-1H-indazole-6-carboxylate(156.3 mg, 0.421 mmol) was dissolved in methanol (1600 μl) andtetrahydrofuran (2500 μl). 1M sodium hydroxide (1684 μA 1.684 mmol) wasadded. The reaction mixture was heated to 50° C. and allowed to stir for0.25 h. The reaction was stopped, cooled to room temperature, quenchedby addition of 1M hydrochloric acid (ca 1.7 mL) till pH<3, and thesolvent evaporated under reduced pressure. The title compound (236.8 mg,0.394 mmol, 94% yield, with 4 equivalents of sodium chloride) recoveredas a white solid. LC-MS: [M+1]=331.1.

Step B:3-Bromo-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1-(4-methylphenyl)-1H-indazole-6-carboxamide

3-Bromo-1-(4-methylphenyl)-1H-indazole-6-carboxylic acid (233.7 mg,0.389 mmol, with 4 equivalents of sodium chloride),(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethanamine bis-hydrochloride (94.2mg, 0.471 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (156.1 mg, 0.814 mmol), 1-hydroxy-7-azabenzotriazole (28.9mg, 0.212 mmol), and N-methylmorpholine (0.171 mL, 1.554 mmol) weredissolved in N,N-dimethylformamide (3.800 mL) at 25° C. The reactionmixture was heated to 50° C. and allowed to stir for 10 min. Thereaction was stopped, quenched by addition of water (ca 0.5 mL) andtrifluoroacetic acid (ca 0.5 mL). The reaction mixture purified directlyby preparative HPLC (Reverse phase (C-18)), eluting withacetonitrile/water+0.1% trifluoroacetic acid. The product fractions wereconcentrated under reduced pressure. The residue was taken up in ethylacetate (15 mL), washed with sodium bicarbonate (2×20 mL) and saturatedaqueous sodium chloride (1×15 mL), dried (sodium sulfate), filtered, andthe solvent evaporated under reduced pressure to give the title compound(153.9 mg, 0.350 mmol, 90% yield) as a tan solid. HRMS: [M+1]=440.0730.

Step C:N-[(1R)-1-(3-Methyl-1,2,4-oxadiazol-5-yl)ethyl]-1-(4-methylphenyl)-3-(prop-1-en-2-yl)-1H-indazole-6-carboxamide

3-Bromo-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1-(4-methylphenyl)-H-indazole-6-carboxamide(155.2 mg, 0.352 mmol),2-isopropenyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (99 μl, 0.529mmol), diisopropylamine (99 μl, 0.705 mmol),triphenylphosphine-3,3′,3″-trisulfonic acid trisodium salt hydrate (24.3mg, 0.038 mmol), and palladium(II) acetate (4.2 mg, 0.019 mmol) weredissolved in N,N-dimethylformamide (1300 μl)/water (400 μl), placed in asealed tube and heated to 100° C. for 1.5 h. The reaction was stopped,cooled to room temperature, quenched by addition of saturated aqueousammonium chloride (10 mL), and extracted with ethyl acetate (3×10 mL).The combined organic phases were washed with saturated aqueous ammoniumchloride (2×10 mL) and saturated aqueous sodium chloride (1×10 mL),dried (sodium sulfate), filtered, and the solvent evaporated underreduced pressure. The crude product was purified by flash chromatography(RediSep SiO₂, 24 g column) on a CombiFlash purification system elutingwith ethyl acetate-hexanes (0-60%). The title compound (102.8 mg, 0.256mmol, 72.6% yield) was recovered as a light yellow/white solid. LC-MS:[M+1]=402.3.

Step D:3-(1,2-Dihydroxypropan-2-yl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1-(4-methylphenyl)-1H-indazole-6-carboxamide

N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1-(4-methylphenyl)-3-(prop-1-en-2-yl)-1H-indazole-6-carboxamide(40.6 mg, 0.101 mmol) was dissolved in acetone (919 μl)/water (92 μl).N-methylmorpholine oxide (15.2 mg, 0.130 mmol) was added. Osmiumtetroxide (2.5% in n-butanol, 32 μl, 2.55 mmol) was added. The reactionmixture was stirred at room temperature for 2.5 h. The reaction wasstopped, quenched by addition of saturated aqueous sodium sulfite (5mL), and extracted with ethyl acetate (3×10 mL). The combined organicphases were washed with saturated aqueous sodium chloride (1×5 mL),dried (sodium sulfate), filtered, and the solvent evaporated underreduced pressure. The residue was purified by preparative HPLC (reversephase, C-18), eluting with acetonitrile/water+0.1% trifluoroacetic acid.The product fraction was diluted with ethyl acetate (15 mL), washed withsaturated aqueous sodium bicarbonate (2×15 mL) and saturated aqueoussodium chloride (1×10 mL), dried (sodium sulfate), and the solventevaporated under reduced pressure to give the title compound (1:1mixture of diastereomers) (32.9 mg, 0.076 mmol, 74.7% yield) as a whitesolid. HRMS: [M+1]=436.2001. ¹H NMR (500 MHz, CDCl₃): δ 8.15 (s, 1H);8.00 (dd, J=8.5, 4.7 Hz, 1H); 7.53-7.46 (m, 3H); 7.30 (d, J=7.9 Hz, 2H);7.13 (d, J=8.0 Hz, 1H); 5.58 (t, J=7.3 Hz, 1H); 4.22 (dd, J=11.3, 3.3Hz, 1H); 3.78 (d, J=11.4 Hz, 1H); 2.42 (s, 3H); 2.45-2.28 (m, 3H);1.71-1.65 (m, 6H).

Example 3.251

3-(2-Hydroxypropan-2-yl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1-(thiophen-3-yl)-1H-indazole-6-carboxamideStep A: Methyl 3-bromo-1H-indazole-6-carboxylate

6-(1H)-indazole carboxylic acid methyl ester (1.5688 g, 8.90 mmol) andcesium carbonate (4.38 g, 13.44 mmol) were dissolved in acetonitrile (89ml) at 25° C. Bromine (0.554 ml, 10.75 mmol) was added and the reactionmixture was allowed to stir for 20 min. The reaction was stopped,quenched by addition of saturated aqueous sodium hydrogen carbonate (30mL) and 10% aqueous sodium thiosulfate (30 mL), and the mixtureextracted with ethyl acetate (3×50 mL). The combined organic phases werewashed with saturated aqueous sodium chloride (1×50 mL), dried (sodiumsulfate), filtered, and the solvent evaporated under reduced pressure.The title compound (2.241 g, 8.79 mmol, 99% yield) was recovered as alight orange/white solid. LC-MS: [M+1]=255.2.

Step B: Methyl 3-bromo-1-(thiophen-3-yl)-1H-indazole-6-carboxylate

Methyl 3-bromo-1H-indazole-6-carboxylate (1.801 g, 7.06 mmol),3-bromothiophene (2.00 mL, 21.35 mmol),trans-1,2-bis(methylamino)cyclohexane (233 μL, 1.478 mmol), copper(I)iodide (142.8 mg, 0.750 mmol), and potassium phosphate tribasic (3.10 g,14.60 mmol) were dissolved in toluene (35 ml) in a sealed tube andheated to 120° C. The reaction was allowed to stir for 3 h. The reactionwas stopped, cooled to room temperature, quenched by addition ofsaturated aqueous ammonium chloride (25 mL), and the mixture extractedwith ethyl acetate (3×30 mL). The combined organic phases were washedwith saturated aqueous sodium chloride (1×20 mL), dried (sodiumsulfate), filtered, and the solvent evaporated under reduced pressure.The crude product was purified by flash chromatography (RediSep SiO₂,120 g column) on a CombiFlash Rf purification system eluting with ethylacetate-hexanes (0-35%). The title compound (1.4177 g, 4.20 mmol, 59.5%yield) was recovered as a white solid. LC-MS: [M+1]=337.0.

Step C: Methyl3-(prop-1-en-2-yl)-1-(thiophen-3-yl)-1H-indazole-6-carboxylate

Methyl 3-bromo-1-(thiophen-3-yl)-1H-indazole-6-carboxylate (1.414 g,4.19 mmol), 2-isopropenyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.58ml, 8.41 mmol), diisopropylamine (1.18 ml, 8.40 mmol),triphenylphosphine-3,3′,3″-trisulfonic acid trisodium salt hydrate(275.8 mg, 0.431 mmol), and palladium(II) acetate (48.3 mg, 0.215 mmol)were dissolved in N,N-dimethylformamide (15 ml)/water (5 mL), placed ina sealed tube and heated to 100° C. for 4 h. The reaction was stopped,cooled to room temperature, quenched by addition of saturated aqueousammonium chloride (20 mL), and the mixture extracted with ethyl acetate(3×20 mL). The combined organic phases were washed with saturatedaqueous sodium chloride (1×15 mL), dried (sodium sulfate), filtered, andthe solvent evaporated under reduced pressure. The crude product waspurified by flash chromatography (RediSep SiO₂, 330 g column) on aCombiFlash Rf purification system eluting with ethyl acetate-hexanes(0-15%). The title compound (1.0847 g, 3.64 mmol, 87% yield) wasrecovered as a white solid. LC-MS: [M+1]=299.2.

Step D: Methyl3-(2-hydroxypropan-2-yl)-1-(thiophen-3-yl)-1H-indazole-6-carboxylate

Methyl 3-(prop-1-en-2-yl)-1-(thiophen-3-yl)-1H-indazole-6-carboxylate(1.0763 g, 3.61 mmol) was dissolved in dimethoxyethane (90 ml)/MeOH (90ml) at 25° C. Cobalt(II) meso-tetraphenylporphine (25.6 mg, 0.038 mmol)and tetraethylammonium borohydride (1.316 g, 9.07 mmol) were addedsequentially. The reaction mixture was allowed to stir for 1.25 h. Thereaction was stopped, quenched by addition of saturated aqueous ammoniumchloride (100 mL), and the mixture extracted with ethyl acetate (3×80mL). The combined organic phases were washed with saturated aqueoussodium chloride (1×80 mL), dried (sodium sulfate), filtered, and thesolvent evaporated under reduced pressure. The crude product waspurified by flash chromatography (RediSep SiO₂, 330 g column) on aCombiFlash Rf purification system eluting with ethyl acetate-hexanes(0-45%). The title compound (912.2 mg, 2.88 mmol, 80% yield) wasrecovered as an orange solid. LC-MS: [M+1]=317.1.

Step E:3-(2-Hydroxypropan-2-yl)-1-(thiophen-3-yl)-1H-indazole-6-carboxylic acid

Methyl3-(2-hydroxypropan-2-yl)-1-(thiophen-3-yl)-1H-indazole-6-carboxylate(909.8 mg, 2.88 mmol) was dissolved in tetrahydrofuran (17.25 mL) andmethanol (11.50 mL) at 25° C. 1M sodium hydroxide (11.50 mL, 11.50 mmol)was added and the reaction mixture heated to 50° C. The reaction mixturewas allowed to stir for 15 min. The reaction was stopped, cooled to roomtemperature, quenched by addition of concentrated hydrochloric acid(0.950 mL), and concentrated under reduced pressure. The title compound(804.8 mg, 2.66 mmol, 93% yield) was recovered as a light orange/whitesolid. LC-MS: [M+1]=303.2.

Step F:3-(2-Hydroxypropan-2-yl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1-(thiophen-3-yl)-1H-indazole-6-carboxamide

3-(2-Hydroxypropan-2-yl)-1-(thiophen-3-yl)-1H-indazole-6-carboxylic acid(457.8 mg, 1.514 mmol), (1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethanaminebis-hydrochloride (374.3 mg, 1.871 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (600.2 mg,3.13 mmol), 1-hydroxy-7-azabenzotriazole (105.3 mg, 0.774 mmol), andN-methylmorpholine (670 μl, 6.09 mmol) were dissolved inN,N-dimethylformamide (15 mL) at 25° C. The reaction mixture was heatedto 50° C. and allowed to stir for 10 min. The reaction was stopped,quenched by addition of water (ca 1 mL) and trifluoroacetic acid (ca 1mL). The reaction mixture was purified directly by preparative HPLC(reverse phase (C-18)), eluting with acetonitrile/water 0.1%trifluoracetic acid, to give the title compound (559.5 mg, 1.360 mmol,90% yield) as a white solid. FIRMS: [M+1]=412.1452. ¹H NMR (400 MHz,CDCl₃): δ 8.22 (s, 1H); 8.06 (d, J=8.5 Hz, 1H); 7.54 (dd, J=8.5, 1.4 Hz,1H); 7.52-7.46 (m, 2H); 6.89 (d, J=7.7 Hz, 1H); 5.65-5.56 (m, 1H); 2.83(s, 1H); 2.40 (s, 3H); 1.81 (s, 6H); 1.73 (d, J=7.1 Hz, 3H).

Example 3.272

N-[(1R)-1-(3-Methyl-1,2,4-oxadiazol-5-yl)ethyl]-3-(morpholin-4-yl)-1-(thiophen-3-yl)-1H-indazole-6-carboxamideStep A: 3-(Morpholin-4-yl)-1-(thiophen-3-yl)-1H-indazole-6-carboxylicacid

To a solution of methyl3-bromo-1-(thiophen-3-yl)-1H-indazole-6-carboxylate (50 mg, 0.148 mmol),morpholine (25.8 μL, 0.297 mmol),tris(dibenzylideneacetone)dipalladium(0) (6.8 mg, 7.41 μmol), and2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (17.7 mg 37.0μmol) in tert-butanol (1.0 mL) was added sodium tert-butoxide (50.0 mg,0.519 mmol). The mixture was sealed in a microwave vial and wasmicrowaved at 130° C. for 5 min. The mixture was cooled to ambienttemperature, and it was filtered through a syringe filter. Filtrate wasconcentrated. Purification by reverse phase HPLC (C-18, 95%water/acetonitrile→5% water/acetonitrile with 0.1% trifluoroacetic acid)gave the title compound as the trifluoroacetate salt (20 mg). LC-MS[M+1] found=330.2.

Step B:N-[(1R)-1-(3-Methyl-1,2,4-oxadiazol-5-yl)ethyl]-3-(morpholin-4-yl)-1-(thiophen-3-yl)-1H-indazole-6-carboxamide

To a solution of3-(morpholin-4-yl)-1-(thiophen-3-yl)-1H-indazole-6-carboxylic acid (20.0mg, 45.0 μmol), (1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethanaminehydrochloride (18.1 mg, 90 μmol, and N-methylmorpholine (24.8 μL, 226μmol) in N,N-dimethylformamide (226 μL) were addedN-[2-(dimethylamino)ethyl]-N-ethylcarbodiimide hydrochloride (15.1 mg,79.0 μmol), 1-hydroxy-7-azabenzotriazole (3.1 mg, 23 pawl). The mixturewas stirred at 50° C. for 1 h. Purification by reverse phase HPLC (C-18,95% water/acetonitrile→5% water/acetonitrile with 0.05% ammoniumhydroxide) gave the title compound (19 mg). HRMS [M+1] found=439.1545.¹H NMR (400 MHz, DMSO-d₆) δ: 9.37 (1H, d, J=7.33 Hz), 8.20 (1H, s), 8.05(1H, d, 8.57 Hz), 7.77-7.74 (2H, m), 7.64 (1H, dd, J=8.56, 1.33 Hz),7.56 (1H, 4.58, 2.12 Hz), 5.43-5.37 (1H, m), 3.83 (4H, t, J=4.39 Hz),3.43 (4H, t, J=4.39 Hz), 2.33 (3H, s), 1.64 (3H, d, J=7.15 Hz).

Example 4.1

3-(2-Chlorophenyl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1-(4-methylphenyl)-1H-indazole-5-carboxamideStep A: Methyl 1-(4-methylphenyl)-1H-indazole-5-carboxylate

5-(1H)indazole carboxylic acid methyl ester (302.1 mg, 1.715 mmol),4-iodotoluene (457.2 mg, 2.097 mmol), copper iodide (17.4 mg, 0.091mmol), trans-1,2-bis(methylamino)cyclohexane (275 μl, 0.348 mmol), andtribasic potassium phosphate (771.4 mg, 3.63 mmol) were dissolved intoluene (1700 μl) at 25° C. under Ar. The reaction mixture was warmed to120° C. and allowed to stir for 14 h. The reaction was stopped, cooledto room temperature, quenched by addition of saturated aqueous ammoniumchloride (10 mL), and extracted with ethyl acetate (3×10 mL). Thecombined organic phases were washed with saturated aqueous sodiumchloride (1×10 mL), dried (sodium sulfate), filtered, and the solventevaporated under reduced pressure. The crude product was purified byflash chromatography (RediSep SiO₂, 40 g column) on a CombiFlash Rfpurification system eluting with ethyl acetate-hexanes (0-65%). Thetitle compound (330.5 mg, 1.241 mmol, 72.4% yield) was recovered as alight yellow/white solid. LC-MS: [M+1]=267.3.

Step B: Methyl 3-bromo-1-(4-methylphenyl)-1H-indazole-5-carboxylate

Methyl 1-(4-methylphenyl)-1H-indazole-5-carboxylate (327.1 mg, 1.228mmol) was dissolved in acetonitrile (12.300 mL) at 25° C. Bromine (158μL, 3.0575 mmol) was added dropwise and the reaction mixture was allowedto stir for 30 min. The reaction was stopped, quenched by addition ofsaturated aqueous sodium hydrogen carbonate (20 mL), and extracted withethyl acetate (3×20 mL). The combined organic phases were washed withsaturated aqueous sodium chloride (1×20 mL), dried (sodium sulfate),filtered, and the solvent evaporated under reduced pressure. The crudeproduct was purified by flash chromatography (RediSep SiO₂, 40 g column)on a CombiFlash Rf purification system eluting with ethylacetate-hexanes (0-45%). The title compound (407.3 mg, 1.180 mmol, 96%yield) was recovered as a light yellow/white solid. LC-MS: [M+1]=346.1.

Step C: methyl3-(2-chlorophenyl)-1-(4-methylphenyl)-1H-indazole-5-carboxylate

Methyl 3-bromo-1-(4-methylphenyl)-1H-indazole-5-carboxylate (150.3 mg,0.435 mmol), 2-chlorophenylboronic acid, pinacol ester (210.4 mg, 0.882mmol), palladium(II) acetate (5.2 mg, 0.023 mmol), copper(I) chloride(88.5 mg, 0.894 mmol), cesium carbonate (431.8 mg, 1.325 mmol), and1,1′-bisdiphenylphosphino ferrocene (26.4 mg, 0.048 mmol) were dissolvedin N,N-dimethylformamide (2200 μl) at 25° C. under Ar. The reactionmixture was warmed to 100° C. and allowed to stir for 3 h. The reactionwas stopped, cooled to room temperature, quenched by addition ofsaturated aqueous ammonium chloride (10 mL), and extracted with ethylacetate (3×10 mL). The combined organic phases washed with saturatedaqueous ammonium chloride (2×10 mL) and saturated aqueous sodiumchloride (1×10 mL), dried (sodium sulfate), filtered, and the solventevaporated under reduced pressure. Crude product purified by flashchromatography (RediSep SiO₂, 24 g column) on a CombiFlash Rfpurification system eluting with ethyl acetate-hexanes (0-30%). Thetitle compound (117.4 mg, 0.312 mmol, 71.6% yield) was recovered as alight yellow/white solid. LC-MS: [M+1]=377.1.

Step D: 3-(2-Chlorophenyl)-1-(4-methylphenyl)-1H-indazole-5-carboxylateacid

Methyl 3-(2-chlorophenyl)-1-(4-methylphenyl)-1H-indazole-5-carboxylate(115.4 mg, 0.306 mmol) was dissolved in tetrahydrofuran (1837 μl) andmethanol (1225 μl) at 25° C. 1 M sodium hydroxide (1225 μl, 1.225 mmol)was added and the reaction mixture heated to 50° C. for 25 min. Thereaction was stopped, cooled to room temperature, quenched by additionof 1M hydrochloric acid (1.225 mL), and concentrated under reducedpressure. The title compound (171.78 mg, 0.257 mmol, 84% yield, with 4equivalents of sodium chloride) was recovered as a white solid. LC-MS:[M+1]=363.2.

Step E:3-(2-(Chlorophenyl)-N-[(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethyl]-1-(4-methylphenyl)-1H-indazole-5-carboxamide

3-(2-Chlorophenyl)-1-(4-methylphenyl)-1H-indazole-5-carboxylic acid(30.6 mg, 0.046 mmol, with 4 equivalents of sodium chloride),(1R)-1-(3-methyl-1,2,4-oxadiazol-5-yl)ethanamine bis-hydrochloride(13.72 mg, 0.069 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (17.52 mg, 0.091 mmol), 1-hydroxy-7-azabenzotriazole (3.11mg, 0.023 mmol), and N-methylmorpholine (20.10 μl, 0.1.83 mmol) weredissolved in N,N-dimethylformamide (457 μl) at 25° C. The reactionmixture was heated to 50° C. and allowed to stir for 10 min. Thereaction was stopped, quenched by addition of water (ca 0.2 mL) andtrifluoroacetic acid (ea 0.2 mL). The reaction mixture was purifieddirectly by preparative HPLC (Reverse phase (C-18)), eluting withacetonitrile/water 0.1% trifluoroacetic acid, to give the title compound(16.2 mg, 0.023 mmol, 50.6% yield) as a white solid. HRMS[M+1]=472.1532. ¹H NMR (400 MHz, CDCl₃): δ 8.17 (s, 1H); 7.88 (dd,J=8.9, 1.7 Hz, 1H); 7.74 (d, J=8.9 Hz, 1H); 7.65-7.60 (m, 3H); 7.56-7.53(m, 1H); 7.42-7.38 (m, 2H); 7.34 (d, J=8.1 Hz, 2H); 6.79 (d, J=7.7 Hz,1H); 5.58 (t, J=7.3 Hz, 1H); 2.42 (s, 3H); 2.36 (s, 3H); 1.68 (d, J=7.1Hz, 3H).

Example 5.4

4-(4-Fluorophenyl)-1-methyl-N-{(1R)-1-[1-oxido-6-(trifluoromethyl)pyridin-3-yl]ethyl}-1-1H-indole-6-carboxamideStep A: Methyl 4-(4-fluorophenyl)-1-methyl-1H-indole-6-carboxylate

To a mixture of methyl 4-bromo-1-methyl-1H-indole-6-carboxylate (0.45 g,1.69 mmol), (4-fluorophenyl)-boronic acid (0.36 g, 2.53 mmol),3,3′,3″-phosphinidynetris (benzensulfonic acid) trisodium salt (81.0 mg,0.13 mmol), palladium(II) acetate (9.5 mg, 0.04 mmol) anddiisopropylamine (0.60 mL, 4.22 mmol) were added N,N-dimethylformamide(6.3 mL) and water (2.1 mL). The mixture was heated to 80° C. for 1 h.The mixture was cooled to ambient temperature and ethyl acetate wasadded. The organic layer was washed with water (3×10 mL), saturatedaqueous sodium chloride (1×10 mL), dried over magnesium sulfate,filtered and concentrated under reduced pressure. Purification by silicagel chromatography (100% hexanes→50% hexanes/ethyl acetate) gave thetitle compound (0.41 g): LC-MS [M+1]=284.1.

Step 13: 4-(4-Fluorophenyl)-1-methyl-1H-indole-6-carboxylic acid

To a solution of methyl4-(4-fluorophenyl)-1-methyl-1H-indole-6-carboxylate (0.41 mg, 1.45 mmol)in methanol (4.8 mL) was added sodium hydroxide (1.0 M; 4.36 mL, 4.36mmol). The mixture was stirred at 45° C. After 18 h, the mixture wascooled to ambient temperature and hydrochloric acid (6.0 M; 0.73 mL,4.36 mmol) was added. The mixture was concentrated to dryness to givethe sodium salt of the title compound (0.61 g): LC-MS [M+1]=270.1

Step C: 4-(4-Fluorophenyl)-1-methyl-N-{(1R)1-[1-oxido-6-(trifluoromethyl)pyridin-3-yl]ethyl}-1H-indole-6-carboxamide

To a solution of sodium salt of4-(4-fluorophenyl)-1-methyl-1H-indole-6-carboxylic acid (25.0 mg, 56.0μmol), hydrochloride salt of(1R)-1-[1-oxido-6-(trifluoromethyl)pyridin-3-yl]ethanamine (16.4 mg,67.0 μmol) and N-methylmorpholine (30.9 μL, 0.28 mmol) inN,N-dimethylformamide (0.56 mL) were added1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (18.9 mg,98.0 μmol), 1-hydroxy-7-azabenzotriazole (3.8 mg, 28.1 μmol). Themixture was stirred at ambient temperature. After 18 h, the mixture wasconcentrated. Purification by reverse phase HPLC (C-18, 95%water/acetonitrile→5% water/acetonitrile with 0.1% NH₄OH) gave the titlecompound (22.1 mg): HRMS [M+1] found=458.1491; ¹H NMR (400 MHz, DMSO): δ8.90 (d, J=7.3 Hz, 1H); 8.53 (s, 1H); 8.06 (s, 1H); 7.94 (d, J=8.4 Hz,1H); 7.74 (dd, =8.4, 5.5 Hz, 2H); 7.69 (s, 1H); 7.59 (d, J=3.1 Hz, 1H);7.55 (d, J=8.4 Hz, 1H); 7.40-7.31 (m, 2H); 6.56 (d, J=3.1 Hz, 1H);5.27-5.18 (m, 1H); 3.90 (s, 3H); 1.56 (d, J=7.1 Hz, 3H).

Example 6.13

3-(2-Chlorophenyl)-1-(4-methylphenyl)-N-{(1R)-1-[1-oxido-6-(trifluoromethyl)pyridin-3-yl]ethyl}-1H-indole-6-carboxamideStep A: Methyl 1-(4-methylphenyl)-1H-indole-6-carboxylate

To a mixture of methyl indole-6-carboxylate (2.03 g, 11.6 mmol),4-bromotoluene (3.56 g, 20.8 mmol),trans-(1R,2R)—N,N′-bismethyl-1,2-cyclohexane diamine (0.18 mL, 1.14mmol), copper iodide (0.12 g, 0.60 mmol) and tribasic potassiumphosphate (5.50 g, 25.9 mmol) was added toluene (11.5 mL). The mixturewas heated in a sealed tube at 180° C. for 10 min. The mixture wascooled to ambient temperature, filtered with Celite and concentratedunder reduced pressure. Purification by silica gel chromatography (100%hexanes→75% hexanes/ethyl acetate) gave the title compound (1.48 g):LC-MS [M+1]=266.1.

Step B: 1-[3-Bromo-1-(4-methylphenyl)-1H-indol-6-yl]ethanone

To a solution of methyl 1-(4-methylphenyl)-1H-indole-6-carboxylate (2.9g, 10.9 mmol) in dichloroethane (105 mL) under Ar was added copper(II)bromide (4.89 g, 21.9 mmol) and sodium hydroxide (1.34 g, 33.5 mmol) andsilica gel (2.1 g). The mixture was heated to 75° C. After 2 h, themixture was cooled to ambient temperature, filtered with Celite andconcentrated under reduced pressure. Purification by silica gelchromatography (100% hexanes→75% hexanes/ethyl acetate) gave the titlecompound (3.28 g): LC-MS [M+1]=344.0.

Step C: Methyl3-(2-chlorophenyl)-1-(4-methylphenyl)-1H-indole-6-carboxylate

To a mixture of 1-[3-bromo-1-(4-methylphenyl)-1H-indol-6-yl]ethanone(0.11 g, 0.31 mmol), 2-chlorophenylboronic acid (73.9 mg, 0.47 mmol),cesium carbonate (0.31 g, 0.94 mmol), copper(I) chloride (32.8 mg, 0.33mmol), 1,1′-bis(diphenylphosphino)ferrocene (18.6 mg, 34.0 μmol) andpalladium(II) acetate (3.5 mg, 16.0 μmol) under Ar was addedN,N-dimethylformamide (4.3 mL). The mixture was stirred in a sealed tubeat 90° C. for 30 min. The mixture was cooled to ambient temperature andquenched with saturated aqueous sodium bicarbonate (1×15 mL). Themixture was extracted with ethyl acetate (3×15 mL). The combined organiclayer was washed with saturated aqueous sodium chloride (1×10 mL), driedover sodium sulfate, filtered and concentrated under reduced pressure.Purification by silica gel chromatography (100% hexanes→95%hexanes/ethyl acetate) gave the title compound (0.11 g): LC-MS[M+1]=376.0.

Step D: 3-(2-Chlorophenyl)-1-(4-methylphenyl)-1H-indole-6-carboxylicacid

To a solution of methyl3-(2-chlorophenyl)-1-(4-methylphenyl)-1H-indole-6-carboxylate (0.11 g,0.28 mmol) in tetrahydrofuran (2.8 mL) was added sodium hydroxide (2.0M; 1.40 mL, 2.80 mmol). Water (1.5 mL) was added and the mixture wasstirred at 50° C. After 18 h, the mixture was cooled to ambienttemperature and 1.0 M hydrochloric acid was added until pH<3. Themixture was extracted with ethyl acetate (3×10 mL). The combined organiclayer was washed with saturated aqueous sodium chloride (1×10 mL), driedover sodium sulfate, filtered and concentrated under reduced pressure togive the title compound (98 mg): LC-MS [M+1]=362.1.

Step E:3-(2-Chlorophenyl)-1-(4-methylphenyl)-N-{(1R)-1-[1-oxido-6-(trifluoromethyl)pyridin-3-yl]ethyl}-1H-indole-6-carboxamide

To a solution of3-(2-chlorophenyl)-1-(4-methylphenyl)-1H-indole-6-carboxylic acid (9.5mg, 26.0 μmol) in N,N-dimethylformamide (0.5 mL) were addedhydrochloride salt of(1R)-1-[1-oxido-6-(trifluoromethyl)pyridin-3-yl]ethanamine (7.3 mg, 30.0μmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (12.5 mg, 65.0μmol), 1-hydroxy-7-azabenzotriazole (2.2 mg, 16.0 μmol) andN-methylmorpholine (12.0 μL, 0.11 mmol). The mixture was stirred atambient temperature for 30 min. Purification by reverse phase HPLC(C-18, 95% water/acetonitrile→5% water/acetonitrile with 0.1%trifluoroacetic acid) gave the bistrifluoroacetate salt of the titlecompound (9.7 mg): HRMS [M+1] found=550.1505; ¹H NMR (500 MHz, CDCl₃): δ8.50 (s, 1H); 8.08 (s, 1H); 7.67 (t, J=7.7 Hz, 3H); 7.61-7.47 (m, 4H);7.44-7.21 (m, 6H); 6.66 (d, J=6.6 Hz, 1H); 5.31-5.23 (m, 1H); 2.44 (s,3H); 1.64 (dd, J=20.9, 7.1 Hz, 3H).

Assay

In Vivo Rat Visceral Pain Model

Male Sprague-Dawley rats, weighing 150-180 g (max. range perexperiment=40 g) at the beginning of the experiments. Animals will bedelivered to the laboratory at least 5 days before the experimentsduring which time they are acclimatized to laboratory conditions. Ratswill be housed in groups of 4, 5 or 6 in macrolon cages (41×25×14 cm or44×28×19 cm) on wood with free access to food and water until tested (oras indicated otherwise). The animal house will be maintained underartificial lighting (12 hours) between 7.00 and 19.00 in a controlledambient temperature of 21±3° C., and relative humidity maintained at40-70%. Information related to any clinical signs and mortality will bearchived with the study materials.

After overnight food-deprivation, male Sprague-Dawley rats are slightlyanesthetized (isoflurane) and injected with 1% acetic acid into thecolon (1.5 ml) using a cannula of 5 cm in length. After a recoveryperiod of 75 minutes, rats are again slightly anesthetized (isoflurane)and a latex balloon of 1.5 cm in length tightly attached to a catheteris inserted via the anus into the descending colon and rectum.Anesthesia is then immediately discontinued. 15 minutes later, the testsubstance is administered p.o. 60 minutes after administration, theballoon is filled with 1.2 ml of water and the number of abdominalcontractions is counted for 10 minutes.

10 rats are studied per group. The test is performed blind. The testsubstance will be evaluated at 3 doses, and compared with the vehiclegroup. Rats will be euthanized at the end of the experiments by exposureto a mixture of O₂/CO₂ (20%/80%) followed by CO₂. Data will be analyzedby comparing treated groups with vehicle control using Mann Whitney Utests.

In Vivo L5 Spinal Nerve Ligation Model

a. Surgery and Post-Operative Care

For the spinal nerve ligation (SNL) procedure, male Sprague Dawley rats(100-200 g; Harlan) are anesthetized using isoflurane (1-5%;inhalation). Using aseptic technique, a dorsal midline incision is madefrom approximately spinal nerve L3 to S2. A combination of sharp andblunt dissection is used to expose the L6/S1 posterior interarticularprocess. The L6 transverse process is visualized and removed, and the L4and L5 spinal nerves are exposed distal to their emergence from theintervertebral foramina. The L5 nerve is then tightly ligated with 6-0silk suture. The muscle is closed with 4-0 absorbable suture and theskin is closed with wound clips. Postoperative monitoring is carried outto assure that animals are exposed to the least amount of pain aspossible. Animals are housed in pairs on bedding and are monitored (2×)daily for three days post-operatively by Laboratory Animal Resourcestaff and then daily by investigator for any signs of possible distress.

b. Behavioral Testing

Prior to surgery, rats are tested for pre-surgery mechanical hind pawwithdrawal thresholds by applying a series of calibrated von Freyfilaments (0.25-15 g) to the left hind paw and determining the medianwithdrawal threshold using the Dixon “up-down” method (Chaplan et al., JNeurosci Meth 53:55, 1994). Rats are placed in individual plasticchambers on an elevated mesh galvanized steel platform and allowed toacclimate for 60 min. Pre-surgery mechanical hind paw withdrawalthresholds are determined, and rats having a threshold<15 g are excludedfrom the study. Following determination of pre-surgery withdrawalthresholds, rats undergo the SNL procedure described above. Between28-35 days following the surgical procedure, rats are tested forpost-surgery thresholds using the procedure described above, and animalsdisplaying a hind paw withdrawal threshold<4.0 g are consideredallodynic (i.e. mechanical hypersensitivity). Effects of test compoundson SNL-induced mechanical hypersensitivity are determined by dosing thecompound along with a vehicle control group and a group receiving thepositive comparator pregabalin (20 mg/kg, p.o.). Efficacy in the SNLmodel is evaluated by determining the % reversal of mechanicalhypersensitivity using the formula:

${\%\mspace{14mu}{reversal}} = {\frac{\left( {{{post}\text{-}{drug}\mspace{14mu}{threshold}} - {{post}\text{-}{surgery}\mspace{14mu}{threshold}}} \right)}{\left( {{{pre}\text{-}{surgery}\mspace{14mu}{threshold}} - {{post}\text{-}{surgery}\mspace{14mu}{threshold}}} \right)} \times 100}$

At the conclusion of the study, all rats are euthanized using CO₂ andplasma and brain tissue are collected for bioanalytical analysis of drugexposures.

In Vivo Complete Freunds Adjuvant (CFA) Model

Male Sprague Dawley rats (300-400 g; Charles River) receive anintradermal injection of CFA (200 ul, 0.5 mg/ml) into the plantar aspectof the left hind paw and are subsequently returned to their cages wherethey are maintained on soft bedding. 72 hrs following CFA injection ratsare tested for post-CFA mechanical hind paw withdrawal thresholds bywrapping the rat in a towel and placing the hind paw (either left orright) in a modified Randall-Sellito paw pinch apparatus (Stoelting,Wood Dale, Ill.). A plastic bar attached to a lever is placed on thedorsum of the hind paw, and an increasing force is applied to the hindpaw until the rat vocalizes or pulls its hind paw away from the bar. Therat's hind paw withdrawal threshold is recorded at that point. Themechanical stimulus is applied to each hind paw 2 times, and the averagepost-CFA mechanical hind paw withdrawal thresholds are determined forboth the left and right hind paw. Following determination of post-CFAwithdrawal thresholds, rats receive test compound, vehicle, or thepositive comparator naproxen (30 mg/kg, p.o.), and effects of compoundson withdrawal thresholds for the inflamed (CFA) hind paw are determined.Efficacy in the CFA model is evaluated by determining the % reversal ofmechanical hypersensitivity using the formula:

${\%\mspace{14mu}{reversal}} = {\frac{\begin{pmatrix}{{{post}\text{-}{drug}\mspace{14mu}{threshold}_{{left}\mspace{14mu}{hind}\mspace{14mu}{paw}}} -} \\{{post}\text{-}{CFA}\mspace{14mu}{threshold}_{{left}\mspace{14mu}{hind}\mspace{14mu}{paw}}}\end{pmatrix}}{\begin{pmatrix}{{{pre}\text{-}{CFA}\mspace{14mu}{threshold}_{{right}\mspace{14mu}{hind}\mspace{14mu}{paw}}} -} \\{{post}\text{-}{CFA}\mspace{14mu}{threshold}_{{left}\mspace{14mu}{hind}\mspace{14mu}{paw}}}\end{pmatrix}} \times 100}$At the conclusion of the study, all rats are euthanized using CO₂ andplasma and brain tissue are collected for bioanalytical analysis of drugexposures.Cystometry in Normal Healthy RatsFemale Sprague-Dawley rats weighed 250-350 g were housed in atemperature- and light (12-h light/dark cycle)-controlled room, and wereallowed access to food and water ad libitum. The animals wereanesthetized with urethane (1.0 g/kg, i.p.). Supplemental urethane wasgiven if necessarily. A lower abdominal midline incision was made toexpose the bladder, and a polyethylene catheter (FE-50) was insertedinto the bladder dome for recording the intravesical pressure andintravesical infusion of physiological saline at the rate of 0.05ml/min. The intravesical pressure was measured using a pressuretransducer, and signal was recorded using a multiple channel dataacquisition system (Power lab, AD Instruments, Biopac systems, ColoradoSprings, Colo.) at a sampling rate of 10 Hz. After confirming stableinter-micturtion interval and micturition pressure by intravesicalinfusion of saline, the drugs were administered intravenously (0.25ml/kg). Intermicturition interval (functional bladder capacity) andmicturition pressure (maximum intravesical pressure) were obtained frommicturitions prior to dosing (baseline) and between 5 to 30 min afterdosing using Chart program (v5.5.4, AD Instruments), and calculated theratio to baseline.Cystometry in Rat Acetic Acid-Induced Hyper-Reflexia Model

Female Sprague-Dawley rats weighed 250-350 g were housed in atemperature- and light (12-h light/dark cycle)-controlled room, and wereallowed access to food and water ad libitum. The animals wereanesthetized with urethane (1.0 g/kg, i.p.). Supplemental urethane wasgiven if necessarily. A lower abdominal midline incision was made toexpose the bladder, and a polyethylene catheter (PE-50) was insertedinto the bladder dome for recording the intravesical pressure andintravesical infusion at the rate of 0.05 ml/min. The intravesicalpressure was measured using a pressure transducer, and signal wasrecorded using a multiple channel data acquisition system (Power lab, ADInstruments, Biopac systems, Colorado Springs, Colo.) at a sampling rateof 10 Hz. After confirming stable inter-micturtion interval andmicturition pressure by intravesical infusion of saline, 0.25% of aceticacid-saline solution was infused at the same infusion rate. After 30-60min, drugs were intravenously infused using infusion pumps at a rate of10 μl/min. Intermicturition interval (functional bladder capacity) andmicturition pressure (maximum intravesical pressure) were obtained frommicturitions prior to dosing (baseline) and between 30 to 45 min afterstarting drug infusion using Chart program (v5.5.4, AD Instruments), andcalculated the ratio to baseline.

Generation of a Human P2X₃ and P2X_(2/3) Stable Cell Line—Human P2X₃receptor cDNA (Accession number NM_(—)002559) was subcloned as a 5′XhoIand 3′HindIII fragment into the expression vector pcDNA5/FRT(Invitrogen). Human P2X₂ receptor cDNA (Accession number NM_(—)174873)was subcloned as a 5′EcoRI and 3′NotI fragment into the expressionvector pIRESneo2 (BD Biosciences Clontech). The human P2X₃ expressionconstruct was transfected using Lipofectamine 2000 (Invitrogen) intoFlp-in—293 cells (Invitrogen) according to the manufacturer'sdirections. Cells positive for flp-mediated recombination of rhesus P2X₃were selected using 150 μg/ml hygromycin. The stable human P2X₃ cellline was co-transfected with the human P2X₂ expression construct usingLipofectamine 2000 as above and co-transfected cells selected using 100mg/ml hygromycin and 1 mg/ml G418. The stable P2X₃ cell line waspropagated in DMEM, 10% FBS, 100 μg/ml hygromycin, and 100 units/mlpenicillin and 100 μg/ml streptomycin, and maintained at 37° and 95%humidity. The stable P2X_(2/3) cell line was propagated as above withthe addition of 500 μg/ml G418.Intracellular Calcium Measurement to Assess Antagonist Affinity—Afluorescent imaging plate reader (FLIPR; Molecular Devices) was used tomonitor intracellular calcium levels using the calcium-chelating dyeFluo-4 (Molecular Probes). The excitation and emission wavelengths usedto monitor fluorescence were 488 nm and 530 nm, respectively. Cellsexpressing either human P2X₃ or human P2X_(2/3) were plated at a densityof 20,000 cells/well (20 pd/well) in 384-well black-walled platesapproximately 20 hours before beginning the assay. On the day of theassay 20 μl of loading buffer (Hank's balanced salt solution, 2.5 mMCaCl₂, 20 mM HEPES, 0.1% BSA, 2.5 mM probenecid, TR-40, Fluo-4, and 138mM NMDG substituted for NaCl) is added and cells dye-loaded for 60 minin the dark at room temperature. Ten minutes prior to adding agonist,the antagonist was added in a volume of 10 μl and allowed to incubate atroom temperature. During this period fluorescence data is collected at 3sec intervals followed by 10 sec intervals. The agonist, α,β-meATP, isadded at a 6× concentration ([α,β-meATP]_(final)=EC₅₀). Followingagonist addition fluorescence was measured at 5 sec intervals andanalyzed based on the increase in peak relative fluorescence units (RFU)compared to the basal fluorescence. Peak fluorescence was used todetermine the inhibitory effect at each concentration of antagonist bythe following equation:% Inhibition=100*(1−((RFU _((drug)) −RFU _((control)))/(RFU_((DMSO only)) −RFU _((control)))))In vitro Electrophysiological Assay—Cells expressing human P2X₃receptors were grown to a confluence of 65-85% 20 to 32 hours prior toassay. The cells were dissociated with trypsin, centrifuged, andresuspended in bath solution at a cell density of 1×10⁶ cells/ml andloaded onto PatchXpress. The bath solution contained 150 mM NaCl, 4 mMKCl, 2 mM CaCl₂, 1.2 mM MgCl₂, 10 mM HEPES, and 11.1 mM glucose, at pH7.2. The intracellular solution contained either 140 mM K-aspartate, 20mM NaCl, 5 mM HEPES, 10 mM EGTA, at pH 7.2 or 30 mM CsCl, 5 mM HEPES, 10mM EGTA, 120 mM CsF, 5 mM NaF, 2 mM MgCl₂, pH=7.3 with CsOH. Agoniststock solutions were prepared in H₂O and diluted in bath solution priorto use. All antagonists were prepared as 10 mM stock solutions in DMSOand diluted in bath solution prior to use. All experiments wereperformed on cells under the whole-cell patch clamp configuration atroom temperature. Up to 16 individual cells could be patch clampedsimultaneously on the PatchXpress instrument. A baseline response wasestablished by repeated CTP (100 μM; for 2 sec.) followed by antagonistincubation for 2 min. in the absence of CTP. After antagonistpreincubation 100 μM CTP and antagonist were co-administered todetermine the inhibitory effect of the antagonist. These steps were thenrepeated on the same cell with a range of concentrations of theantagonist. A maximum of five concentrations of antagonist were testedon any individual cell. The control P2X₃ current amplitude(I_(P2X3−(control))) was taken as an average of the peak currentamplitude from the last two agonist additions prior to incubation withan antagonist. The peak P2X₃ current amplitude in the presence of anantagonist (I_(P2X3−(drug))) was used to calculate the inhibitory effectat each concentration of the antagonist according to the followingequation:% inhibition of P2X ₃=100*(I _(P2X3−(control)) −I _(P2X3−(drug)))/I_(P2X3−(control))

Each concentration of an antagonist was tested on at least twoindependent cells. The concentration of drug required to inhibit P2X₃current by 50% (IC₅₀) was determined by fitting of the Hill equation tothe averaged % inhibition data at each concentration:% of Control=100·(1+([Drug]/IC ₅₀)^(p))⁻¹In vitro Electrophysiological Assay for P2X_(2/3)—P2X_(2/3) was assayedas above with two protocol modifications: 1) 30 μM α,β-meATP used asagonist; and 2) current amplitude was measured at the end of 2-secondagonist application.

Using the assays described herein the compounds of this invention werefound to be active for the P2X₃ receptor. The compounds of formula Ihave an IC₅₀ activity of 100 μM or less for the P2X₃ receptor. Many ofthe compounds of formula I have an IC₅₀ of less than 200 nM. Forexample, the compounds below have IC₅₀<250 nM in the “IntracellularCalcium Measurement to Assess Antagonist Affinity” assay. In particular,example 1.1 has an IC₅₀=46 nM, example 1.16 has an IC₅₀=100 nM, example1.35 has an IC₅₀=320 nM, example 2.1 has an IC₅₀=63 nM and example 2.3has an IC₅₀=120 nM.

What is claimed:
 1. A compound of structural formula III and VII:

or pharmaceutically acceptable salts and individual enantiomers anddiastereomers thereof wherein: R² represents H, C₁₋₆ alkyl, CF₃, OH; R³is selected from the group consisting of (CHR^(Y))_(n)pyridyl,(CHR^(Y))_(n)oxidopyridyl, (CHR^(Y))_(n)pyrimidinyl,(CHR^(Y))_(n)triazolyl, (CHR^(Y))_(n)phenyl, (CHR^(Y))_(n)pyrazinyl,(CHR^(Y))_(n)pyrazolyl, (CHR^(Y))_(n)oxadiazolyl,(CHR^(Y))_(n)thiazolyl, (CHR^(Y))_(n)thiadiazolyl,(CHR^(Y))_(n)indazopyridyl, C₁₋₆ alkyl, and (CHR^(Y))_(n)C₃₋₆cycloalkyl, wherein R^(Y) represents H, C₁₋₆ alkyl optionallysubstituted with R^(a), CF₃, or OH or R² and R³ can be combined with thenitrogen to which they are attached to form a C₅₋₁₀ heterocyclyloptionally substituted with 1 to 3 groups of R^(a); R⁶ representshydrogen, OR², (CH₂)_(n)CF₃, halogen, C(R²)₂OR², C₂₋₆ alkenyl, C₂₋₆alkynyl, (CH₂)_(n)C₆₋₁₀ aryl, (CH₂)_(n)C₅₋₁₀ heterocyclyl, said alkyl,cycloalkyl, aryl and heterocyclyl optionally substituted with 1 to 3groups of R^(a); R^(6a) is R⁶; R^(a) represents C₁₋₆ alkyl, halogen,hydroxyl, OR² (CH₂)_(n)CF₃, —O—, C₃₋₆ cycloalkyl, NR²C(O)R², C(O)N(R²)₂,C(R²)₂OR², C(O)R², O(CH₂)_(n)C(O)N(R²)₂, O(CH₂)_(n)C(O)OR²,C(O)C₅₋₁₀heterocyclyl, NO₂, CN, N(R²)₂, C(O)OR², SO₂R², OR²,(CH₂)_(n)C₅₋₁₀ heterocyclyl, or (CH₂)_(n)C₆₋₁₀ aryl, said alkyl,heterocyclyl and aryl optionally substituted with 1 to 3 groups of C₁₋₆alkyl, halogen, hydroxyl, (CH₂)_(n)CF₃, or CN; and n represents 0 to 4.2. The compound according to claim 1 wherein R^(6a) is (CH₂)_(n)C₆₋₁₀aryl wherein n is 0, said aryl optionally substituted with 1 to 3 groupsof R^(a).
 3. The compound according to claim 1 wherein R^(6a) is(CH₂)_(n)C₅₋₁₀ heterocyclyl wherein n is 0, said heterocyclyl optionallysubstituted with 1 to 3 groups of R^(a).
 4. The compound according toclaim 1 wherein R^(6a) is optionally substituted phenyl or pyridyl. 5.The compound according to claim 1 wherein R^(6a) is selected from thegroup consisting of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₃₋₁₀ cycloalkyl, saidalkyl, alkenyl and cycloalkyl optionally substituted with 1 to 3 groupsof R^(a).
 6. The compound according to claim 1 wherein R⁶ represents(CH₂)_(n)phenyl, thiophenyl, thiazolyl, furanyl, or pyrimidinyl., all ofwhich are optionally substituted with 1 to 3 groups of R^(a), R² ishydrogen and all of which are optionally substituted with 1 to 3 groupsof R^(a).
 7. The compound according to claim 1 of formula III:

or pharmaceutically acceptable salts and individual enantiomers anddiastereomers thereof.
 8. The compound according to claim 1 of formulaVII:

or pharmaceutically acceptable salts and individual enantiomers anddiastereomers thereof.
 9. A compound represented by Tables 3 and 4 orpharmaceutically acceptable salts and individual enantiomers anddiastereomers thereof.
 10. The compound according to claim 9 which is:

Example R³ B R⁶ 3.1

3.98

3.142

3.208

3.248

3.251

3.257

3.267

3.272

3.280

or pharmaceutically acceptable salts and individual enantiomers anddiastereomers thereof.
 11. A pharmaceutical composition comprising aninert carrier and an effective amount of a compound according to claim1.